Polypeptides having feruloyl esterase activity and polynucleotides encoding same

ABSTRACT

The present invention relates to isolated polypeptides having feruloyl esterase activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

REFERENCE TO A SEQUENCE LISTING

This application contains a Sequence Listing filed electronically byEFS, which is incorporated herein by reference.

REFERENCE TO A DEPOSIT OF BIOLOGICAL MATERIAL

This application contains a reference to a deposit of biologicalmaterial, which deposit is incorporated herein by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to isolated polypeptides having feruloylesterase activity and isolated polynucleotides encoding thepolypeptides. The invention also relates to nucleic acid constructs,vectors, and host cells comprising the polynucleotides as well asmethods of producing and using the polypeptides.

2. Description of the Related Art

Polysaccharides constitute 90% of plant cell walls and can be dividedinto three groups: cellulose, hemicellulose, and pectin. Celluloserepresents the major constituent of cell wall polysaccharides.Hemicelluloses are the second most abundant constituent of plant cellwalls. The major hemicellulose polymer is xylan. The structure of xylansfound in cell walls of plants can differ significantly depending ontheir origin, but they all contain a beta-1,4-linked D-xylose backbone.The beta-1,4-linked D-xylose backbone can be substituted by various sidegroups, such as L-aribinose, D-galactose, acetyl, feruloyl, p-coumaroyl,and glucuronic acid residues.

The biodegradation of the xylan backbone depends on two classes ofenzymes: endoxylanases and beta-xylosidases. Endoxylanases (EC 3.2.1.8)cleave the xylan backbone into smaller oligosaccharides, which can befurther degraded to xylose by beta-xylosidases (EC 3.2.1.37). Otherenzymes involved in the degradation of xylan include, for example,acetylxylan esterase, arabinase, alpha-glucuronidase, feruloyl esterase,and p-coumaric acid esterase.

Faulds and Williamson, 1991, J. Gen. Microbiol. 137: 2339-2345, describethe purification and characterization of 4-hydroxy-3-methoxy-cinnamic(ferulic) acid esterase from Streptomyces olivochromogenes. Faulds andWilliamson, 1994, Microbiology 140: 779-787, describe the purificationand characterization of a feruloyl esterase from Aspergillus niger.Kroon et al., 1996, Biotechnol. Appl. Biochem. 23: 255-262, describe thepurification and characterisation of a novel feruloyl esterase inducedby growth of Aspergillus niger on sugarbeet pulp. deVries et al., 1997,Appl. Environ. Microbiol. 63: 4638-4644, disclose feruloyl esterasegenes from Aspergillus niger and Aspergillus tubingensis. Castanares etal., 1992, Enzyme Microbiol. Technol. 14: 875-884, describe thepurification and properties of a feruloyl/p-coumaroyl esterase from thefungus Penicillium pinophilum.

The present invention relates to polypeptides having feruloyl esteraseactivity and polynucleotides encoding the polypeptides.

SUMMARY OF THE INVENTION

The present invention relates to isolated polypeptides having feruloylesterase activity selected from the group consisting of:

(a) a polypeptide comprising an amino acid sequence having at least 75%sequence identity to the mature polypeptide of SEQ ID NO: 2;

(b) a polypeptide encoded by a polynucleotide that hybridizes under atleast medium-high stringency conditions with (i) the mature polypeptidecoding sequence of SEQ ID NO: 1, (ii) the cDNA sequence contained in themature polypeptide coding sequence of SEQ ID NO: 1, or (iii) afull-length complementary strand of (i) or (ii);

(c) a polypeptide encoded by a polynucleotide comprising a nucleotidesequence having at least 75% sequence identity to the mature polypeptidecoding sequence of SEQ ID NO: 1; and

(d) a variant comprising a substitution, deletion, and/or insertion ofone or more (several) amino acids of the mature polypeptide of SEQ IDNO: 2.

The present invention also relates to isolated polynucleotides encodingpolypeptides having feruloyl esterase activity, selected from the groupconsisting of:

(a) a polynucleotide encoding a polypeptide comprising an amino acidsequence having at least 75% sequence identity to the mature polypeptideof SEQ ID NO: 2;

(b) a polynucleotide that hybridizes under at least medium-highstringency conditions with (i) the mature polypeptide coding sequence ofSEQ ID NO: 1, (ii) the cDNA sequence contained in the mature polypeptidecoding sequence of SEQ ID NO: 1, or (iii) a full-length complementarystrand of (i) or (ii);

(c) a polynucleotide comprising a nucleotide sequence having at least75% sequence identity to the mature polypeptide coding sequence of SEQID NO: 1; and

(d) a polynucleotide encoding a variant comprising a substitution,deletion, and/or insertion of one or more (several) amino acids of themature polypeptide of SEQ ID NO: 2.

The present invention also relates to nucleic acid constructs,recombinant expression vectors, and recombinant host cells comprisingthe polynucleotides, and to methods of producing the polypeptides havingferuloyl esterase activity.

The present invention also relates to methods of inhibiting theexpression of a polypeptide having feruloyl esterase activity in a cell,comprising administering to the cell or expressing in the cell adouble-stranded RNA (dsRNA) molecule, wherein the dsRNA comprises asubsequence of a polynucleotide of the present invention. The presentalso relates to such a double-stranded inhibitory RNA (dsRNA) molecule,wherein optionally the dsRNA is a siRNA or a miRNA molecule.

The present invention also relates to methods of using the polypeptideshaving feruloyl esterase activity for the degradation or conversion ofcellulosic or xylan-containing material.

The present invention also relates to plants comprising an isolatedpolynucleotide encoding a polypeptide having feruloyl esterase activity.

The present invention also relates to methods of producing a polypeptidehaving feruloyl esterase activity, comprising: (a) cultivating atransgenic plant or a plant cell comprising a polynucleotide encodingthe polypeptide having feruloyl esterase activity under conditionsconducive for production of the polypeptide; and (b) recovering thepolypeptide.

The present invention also relates to an isolated polynucleotideencoding a signal peptide comprising or consisting of amino acids 1 to25 of SEQ ID NO: 2, an isolated polynucleotide encoding a propeptidecomprising or consisting of amino acids 26 to 585 of SEQ ID NO: 2, andan isolated polynucleotide encoding a signal peptide and a propeptidecomprising or consisting of amino acids 1 to 25 of SEQ ID NO: 2; tonucleic acid constructs, expression vectors, and recombinant host cellscomprising the polynucleotides; and to methods of producing a protein.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the genomic DNA sequence and the deduced amino acidsequence of a Thielavia terrestris NRRL 8126 feruloyl esterase (SEQ IDNOs: 1 and 2, respectively).

FIG. 2 shows a restriction map of pDFng106.

FIG. 3 shows a restriction map of pDFng105.

DEFINITIONS

Feruloyl esterase activity: The term “feruloyl esterase activity” isdefined herein as a 4-hydroxy-3-methoxycinnamoyl-sugar hydrolaseactivity (EC 3.1.1.73) that catalyzes the hydrolysis of the4-hydroxy-3-methoxycinnamoyl (feruloyl) group from an esterified sugar,which is usually arabinose in “natural” substrates, to produce ferulate(4-hydroxy-3-methoxycinnamate). Feruloyl esterase is also known asferulic acid esterase, hydroxycinnamoyl esterase, FAE-III, cinnamoylester hydrolase, FAEA, cinnAE, FAE-I, or FAE-II. A feruloyl esterase ofthe present invention can also have tannase activity (EC 3.1.1.20). Forpurposes of the present invention, feruloyl esterase activity isdetermined using 0.5 mM p-nitrophenylferulate as substrate in 50 mMsodium acetate pH 5.0. One unit of feruloyl esterase activity equals theamount of enzyme capable of releasing 1 μmole of p-nitrophenolate anionper minute at pH 5, 25° C.

The polypeptides of the present invention have at least 20%, preferablyat least 40%, more preferably at least 50%, more preferably at least60%, more preferably at least 70%, more preferably at least 80%, evenmore preferably at least 90%, most preferably at least 95%, and evenmost preferably at least 100% of the feruloyl esterase activity of themature polypeptide of SEQ ID NO: 2.

Cellulolytic activity: The term “cellulolytic activity” is definedherein as a biological activity that hydrolyzes a cellulosic material.The two basic approaches for measuring cellulolytic activity include:(1) measuring the total cellulolytic activity, and (2) measuring theindividual cellulolytic activities (endoglucanases, cellobiohydrolases,and beta-glucosidases) as reviewed in Zhang et al., Outlook forcellulase improvement: Screening and selection strategies, 2006,Biotechnology Advances 24: 452-481. Total cellulolytic activity isusually measured using insoluble substrates, including Whatman No 1filter paper, microcrystalline cellulose, bacterial cellulose, algalcellulose, cotton, pretreated lignocellulose, etc. The most common totalcellulolytic activity assay is the filter paper assay using Whatman No 1filter paper as the substrate. The assay was established by theInternational Union of Pure and Applied Chemistry (IUPAC) (Ghose, 1987,Measurement of cellulase activities, Pure Appl. Chem. 59: 257-68).

For purposes of the present invention, cellulolytic activity isdetermined by measuring the increase in hydrolysis of a cellulosicmaterial by cellulolytic enzyme(s) under the following conditions: 1-20mg of cellulolytic protein/g of cellulose in PCS for 3-7 days at 50-65°C. compared to a control hydrolysis without addition of cellulolyticprotein. Typical conditions are 1 ml reactions, washed or unwashed PCS,5% insoluble solids, 50 mM sodium acetate pH 5, 1 mM MnSO₄, 50-65° C.,72 hours, sugar analysis by AMINEX® HPX-87H column (Bio-RadLaboratories, Inc., Hercules, Calif., USA).

Endoglucanase: The term “endoglucanase” is defined herein as anendo-1,4-(1,3;1,4)-beta-D-glucan 4-glucanohydrolase (E.C. 3.2.1.4),which catalyses endohydrolysis of 1,4-beta-D-glycosidic linkages incellulose, cellulose derivatives (such as carboxymethyl cellulose andhydroxyethyl cellulose), lichenin, beta-1,4 bonds in mixed beta-1,3glucans such as cereal beta-D-glucans or xyloglucans, and other plantmaterial containing cellulosic components. Endoglucanase activity can bedetermined based on a reduction in substrate viscosity or increase inreducing ends determined by a reducing sugar assay (Zhang et al., 2006,Biotechnology Advances 24: 452-481). For purposes of the presentinvention, endoglucanase activity is determined using carboxymethylcellulose (CMC) hydrolysis according to the procedure of Ghose, 1987,Pure and Appl. Chem. 59: 257-268.

Cellobiohydrolase: The term “cellobiohydrolase” is defined herein as a1,4-beta-D-glucan cellobiohydrolase (E.C. 3.2.1.91), which catalyzes thehydrolysis of 1,4-beta-D-glucosidic linkages in cellulose,cellooligosaccharides, or any beta-1,4-linked glucose containingpolymer, releasing cellobiose from the reducing or non-reducing ends ofthe chain (Teeri, 1997, Crystalline cellulose degradation: New insightinto the function of cellobiohydrolases, Trends in Biotechnology 15:160-167; Teeri et al., 1998, Trichoderma reesei cellobiohydrolases: whyso efficient on crystalline cellulose?, Biochem. Soc. Trans. 26:173-178). For purposes of the present invention, cellobiohydrolaseactivity is determined using a fluorescent disaccharide derivative4-methylumbelliferyl-β-D-lactoside according to the procedures describedby van Tilbeurgh et al., 1982, FEBS Letters 149: 152-156 and vanTilbeurgh and Claeyssens, 1985, FEBS Letters 187: 283-288.

Beta-glucosidase: The term “beta-glucosidase” is defined herein as abeta-D-glucoside glucohydrolase (E.C. 3.2.1.21), which catalyzes thehydrolysis of terminal non-reducing beta-D-glucose residues with therelease of beta-D-glucose. For purposes of the present invention,beta-glucosidase activity is determined according to the basic proceduredescribed by Venturi et al., 2002, Extracellular beta-D-glucosidase fromChaetomium thermophilum var. coprophilum: production, purification andsome biochemical properties, J. Basic Microbiol. 42: 55-66. One unit ofbeta-glucosidase activity is defined as 1.0 μmole of p-nitrophenolproduced per minute at 40° C., pH 5 from 1 mMp-nitrophenyl-beta-D-glucopyranoside as substrate in 100 mM sodiumcitrate containing 0.01% TWEEN® 20.

Cellulolytic enhancing activity: The term “cellulolytic enhancingactivity” is defined herein as a biological activity that enhances thehydrolysis of a cellulosic material by polypeptides having cellulolyticactivity. For purposes of the present invention, cellulolytic enhancingactivity is determined by measuring the increase in reducing sugars orin the increase of the total of cellobiose and glucose from thehydrolysis of a cellulosic material by cellulolytic protein under thefollowing conditions: 1-50 mg of total protein/g of cellulose in PCS,wherein total protein is comprised of 50-99.5% w/w cellulolytic proteinand 0.5-50% w/w protein of cellulolytic enhancing activity for 1-7 dayat 50-65° C. compared to a control hydrolysis with equal total proteinloading without cellulolytic enhancing activity (1-50 mg of cellulolyticprotein/g of cellulose in PCS). In a preferred aspect, a mixture ofCELLUCLAST® 1.5L (Novozymes NS, Bagsværd, Denmark) in the presence of 3%of total protein weight Aspergillus oryzae beta-glucosidase(recombinantly produced in Aspergillus oryzae according to WO 02/095014)or 3% of total protein weight Aspergillus fumigatus beta-glucosidase(recombinantly produced in Aspergillus oryzae as described in WO2002/095014) of cellulase protein loading is used as the source of thecellulolytic activity.

The polypeptides having cellulolytic enhancing activity enhance thehydrolysis of a cellulosic material catalyzed by proteins havingcellulolytic activity by reducing the amount of cellulolytic enzymerequired to reach the same degree of hydrolysis preferably at least1.01-fold, more preferably at least 1.05-fold, more preferably at least1.10-fold, more preferably at least 1.25-fold, more preferably at least1.5-fold, more preferably at least 2-fold, more preferably at least3-fold, more preferably at least 4-fold, more preferably at least5-fold, even more preferably at least 10-fold, and most preferably atleast 20-fold.

Xylan degrading activity: The terms “xylan degrading activity” or“xylanolytic activity” are defined herein as a biological activity thathydrolyzes xylan-containing material.

The two basic approaches for measuring xylanolytic activity include: (1)measuring the total xylanolytic activity, and (2) measuring theindividual xylanolytic activities (endoxylanases, beta-xylosidases,arabinofuranosidases, alpha-glucuronidases, acetylxylan esterases,feruloyl esterases, and alpha-glucuronyl esterases). Recent progress inassays of xylanolytic enzymes was summarized in several publicationsincluding Biely and Puchard, Recent progress in the assays ofxylanolytic enzymes, 2006, Journal of the Science of Food andAgriculture 86 (11): 1636-1647; Spanikova and Biely, 2006, Glucuronoylesterase—Novel carbohydrate esterase produced by Schizophyllum commune,FEBS Letters 580 (19): 4597-4601; Herrmann, Vrsanska, Jurickova, Hirsch,Biely, and Kubicek, 1997, The beta-D-xylosidase of Trichoderma reesei isa multifunctional beta-D-xylan xylohydrolase, Biochemical Journal 321:375-381.

Total xylan degrading activity can be measured by determining thereducing sugars formed from various types of xylan, including oat spelt,beechwood, and larchwood xylans, or by photometric determination of dyedxylan fragments released from various covalently dyed xylans. The mostcommon total xylanolytic activity assay is based on production ofreducing sugars from polymeric 4-O-methyl glucuronoxylan as described inBailey, Biely, Poutanen, 1992, Interlaboratory testing of methods forassay of xylanase activity, Journal of Biotechnology 23 (3): 257-270.

For purposes of the present invention, xylan degrading activity isdetermined by measuring the increase in hydrolysis of birchwood xylan(Sigma Chemical Co., Inc., St. Louis, Mo., USA) by xylan-degradingenzyme(s) under the following typical conditions: 1 ml reactions, 5mg/ml substrate (total solids), 5 mg of xylanolytic protein/g ofsubstrate, 50 mM sodium acetate pH 5, 50° C., 24 hours, sugar analysisusing p-hydroxybenzoic acid hydrazide (PHBAH) assay as described byLever, 1972, A new reaction for colorimetric determination ofcarbohydrates, Anal. Biochem 47: 273-279.

Xylanase activity: The term “xylanase activity” is defined herein as a1,4-beta-D-xylan-xylohydrolase activity (E.C. 3.2.1.8) that catalyzesthe endo-hydrolysis of 1,4-beta-D-xylosidic linkages in xylans. Forpurposes of the present invention, xylanase activity is determined usingbirchwood xylan as substrate. One unit of xylanase activity is definedas 1.0 μmole of reducing sugar (measured in glucose equivalents asdescribed by Lever, 1972, A new reaction for colorimetric determinationof carbohydrates, Anal. Biochem 47: 273-279) produced per minute duringthe initial period of hydrolysis at 50° C., pH 5 from 2 g of birchwoodxylan per liter as substrate in 50 mM sodium acetate containing 0.01%TWEEN® 20.

Beta-xylosidase activity: The term “beta-xylosidase activity” is definedherein as a beta-D-xyloside xylohydrolase (E.C. 3.2.1.37) that catalyzesthe exo-hydrolysis of short beta (1→4)-xylooligosaccharides, to removesuccessive D-xylose residues from the non-reducing termini. For purposesof the present invention, one unit of beta-xylosidase activity isdefined as 1.0 μmole of p-nitrophenol produced per minute at 40° C., pH5 from 1 mM p-nitrophenyl-beta-D-xyloside as substrate in 100 mM sodiumcitrate containing 0.01% TWEEN® 20.

Acetylxylan esterase activity: The term “acetylxylan esterase activity”is defined herein as a carboxylesterase activity (EC 3.1.1.72) thatcatalyses the hydrolysis of acetyl groups from polymeric xylan,acetylated xylose, acetylated glucose, alpha-napthyl acetate, andp-nitrophenyl acetate. For purposes of the present invention,acetylxylan esterase activity is determined using 0.5 mMp-nitrophenylacetate as substrate in 50 mM sodium acetate pH 5.0containing 0.01% TWEEN™ 20. One unit of acetylxylan esterase activity isdefined as the amount of enzyme capable of releasing 1 μmole ofp-nitrophenolate anion per minute at pH 5, 25° C.

Alpha-glucuronidase activity: The term “alpha-glucuronidase activity” isdefined herein as an alpha-D-glucosiduronate glucuronohydrolase activity(EC 3.2.1.139) that catalyzes the hydrolysis of an alpha-D-glucuronosideto D-glucuronate and an alcohol. For purposes of the present invention,alpha-glucuronidase activity is determined according to de Vries, 1998,J. Bacteriol. 180: 243-249. One unit of alpha-glucuronidase activityequals the amount of enzyme capable of releasing 1 μmole of glucuronicor 4-O-methylglucuronic acid per minute at pH 5, 40° C.

Alpha-L-arabinofuranosidase activity: The term“alpha-L-arabinofuranosidase activity” is defined herein as analpha-L-arabinofuranoside arabinofuranohydrolase activity (EC 3.2.1.55)that catalyzes the hydrolysis of terminal non-reducingalpha-L-arabinofuranoside residues in alpha-L-arabinosides. The enzymeactivity acts on alpha-L-arabinofuranosides, alpha-L-arabinanscontaining (1,3)- and/or (1,5)-linkages, arabinoxylans, andarabinogalactans. Alpha-L-arabinofuranosidase is also known asarabinosidase, alpha-arabinosidase, alpha-L-arabinosidase,alpha-arabinofuranosidase, polysaccharide alpha-L-arabinofuranosidase,alpha-L-arabinofuranoside hydrolase, L-arabinosidase, oralpha-L-arabinanase. For purposes of the present invention,alpha-L-arabinofuranosidase activity is determined using 5 mg of mediumviscosity wheat arabinoxylan (Megazyme International Ireland, Ltd.,Bray, Co. Wicklow, Ireland) per ml of 100 mM sodium acetate pH 5 in atotal volume of 200 μl for 30 minutes at 40° C. followed by arabinoseanalysis by AMINEX® HPX-87H column chromatography (Bio-Rad Laboratories,Inc., Hercules, Calif., USA).

Cellulosic material: The cellulosic material can be any materialcontaining cellulose. The predominant polysaccharide in the primary cellwall of biomass is cellulose, the second most abundant is hemicellulose,and the third is pectin. The secondary cell wall, produced after thecell has stopped growing, also contains polysaccharides and isstrengthened by polymeric lignin covalently cross-linked tohemicellulose. Cellulose is a homopolymer of anhydrocellobiose and thusa linear beta-(1-4)-D-glucan, while hemicelluloses include a variety ofcompounds, such as xylans, xyloglucans, arabinoxylans, and mannans incomplex branched structures with a spectrum of substituents. Althoughgenerally polymorphous, cellulose is found in plant tissue primarily asan insoluble crystalline matrix of parallel glucan chains.Hemicelluloses usually hydrogen bond to cellulose, as well as to otherhemicelluloses, which help stabilize the cell wall matrix.

Cellulose is generally found, for example, in the stems, leaves, hulls,husks, and cobs of plants or leaves, branches, and wood of trees. Thecellulosic material can be, but is not limited to, herbaceous material,agricultural residue, forestry residue, municipal solid waste, wastepaper, and pulp and paper mill residue (see, for example, Wiselogel etal., 1995, in Handbook on Bioethanol (Charles E. Wyman, editor), pp.105-118, Taylor & Francis, Washington D.C.; Wyman, 1994, BioresourceTechnology 50: 3-16; Lynd, 1990, Applied Biochemistry and Biotechnology24/25: 695-719; Mosier et al., 1999, Recent Progress in Bioconversion ofLignocellulosics, in Advances in Biochemical Engineering/Biotechnology,T. Scheper, managing editor, Volume 65, pp. 23-40, Springer-Verlag, NewYork). It is understood herein that the cellulose may be in the form oflignocellulose, a plant cell wall material containing lignin, cellulose,and hemicellulose in a mixed matrix. In a preferred aspect, thecellulosic material is lignocellulose.

In one aspect, the cellulosic material is herbaceous material. Inanother aspect, the cellulosic material is agricultural residue. Inanother aspect, the cellulosic material is forestry residue. In anotheraspect, the cellulosic material is municipal solid waste. In anotheraspect, the cellulosic material is waste paper. In another aspect, thecellulosic material is pulp and paper mill residue.

In another aspect, the cellulosic material is corn stover. In anotheraspect, the cellulosic material is corn fiber. In another aspect, thecellulosic material is corn cob. In another aspect, the cellulosicmaterial is orange peel. In another aspect, the cellulosic material isrice straw. In another aspect, the cellulosic material is wheat straw.In another aspect, the cellulosic material is switch grass. In anotheraspect, the cellulosic material is miscanthus. In another aspect, thecellulosic material is bagasse.

In another aspect, the cellulosic material is microcrystallinecellulose. In another aspect, the cellulosic material is bacterialcellulose. In another aspect, the cellulosic material is algalcellulose. In another aspect, the cellulosic material is cotton linter.In another aspect, the cellulosic material is amorphous phosphoric-acidtreated cellulose. In another aspect, the cellulosic material is filterpaper.

The cellulosic material may be used as is or may be subjected topretreatment, using conventional methods known in the art, as describedherein. In a preferred aspect, the cellulosic material is pretreated.

Pretreated corn stover: The term “PCS” or “Pretreated Corn Stover” isdefined herein as a cellulosic material derived from corn stover bytreatment with heat and dilute sulfuric acid.

Xylan-containing material: The term “xylan-containing material” isdefined herein as any material comprising a plant cell wallpolysaccharide containing a backbone of beta-(1-4)-linked xyloseresidues. Xylans of terrestrial plants are heteropolymers possessing abeta-(1-4)-D-xylopyranose backbone, which is branched by shortcarbohydrate chains. They comprise D-glucuronic acid or its 4-O-methylether, L-arabinose, and/or various oligosaccharides, composed ofD-xylose, L-arabinose, D- or L-galactose, and D-glucose. Xylan-typepolysaccharides can be divided into homoxylans and heteroxylans, whichinclude glucuronoxylans, (arabino)glucuronoxylans,(glucurono)arabinoxylans, arabinoxylans, and complex heteroxylans. See,for example, Ebringerova et al., 2005, Adv. Polym. Sci. 186: 1-67.

In the methods of the present invention, any material containing xylanmay be used. In a preferred aspect, the xylan-containing material islignocellulose.

Isolated polypeptide: The term “isolated polypeptide” as used hereinrefers to a polypeptide that is isolated from a source. In a preferredaspect, the polypeptide is at least 1% pure, preferably at least 5%pure, more preferably at least 10% pure, more preferably at least 20%pure, more preferably at least 40% pure, more preferably at least 60%pure, even more preferably at least 80% pure, and most preferably atleast 90% pure, as determined by SDS-PAGE.

Substantially pure polypeptide: The term “substantially purepolypeptide” denotes herein a polypeptide preparation that contains atmost 10%, preferably at most 8%, more preferably at most 6%, morepreferably at most 5%, more preferably at most 4%, more preferably atmost 3%, even more preferably at most 2%, most preferably at most 1%,and even most preferably at most 0.5% by weight of other polypeptidematerial with which it is natively or recombinantly associated. It is,therefore, preferred that the substantially pure polypeptide is at least92% pure, preferably at least 94% pure, more preferably at least 95%pure, more preferably at least 96% pure, more preferably at least 97%pure, more preferably at least 98% pure, even more preferably at least99% pure, most preferably at least 99.5% pure, and even most preferably100% pure by weight of the total polypeptide material present in thepreparation. The polypeptides of the present invention are preferably ina substantially pure form, i.e., that the polypeptide preparation isessentially free of other polypeptide material with which it is nativelyor recombinantly associated. This can be accomplished, for example, bypreparing the polypeptide by well-known recombinant methods or byclassical purification methods.

Mature polypeptide: The term “mature polypeptide” is defined herein as apolypeptide in its final form following translation and anypost-translational modifications, such as N-terminal processing,C-terminal truncation, glycosylation, phosphorylation, etc. In oneaspect, the mature polypeptide is amino acids 26 to 585 of SEQ ID NO: 2based on the SignalP program (Nielsen et al., 1997, Protein Engineering10:1-6) program that predicts amino acids 1 to 25 of SEQ ID NO: 2 are asignal peptide.

Mature polypeptide coding sequence: The term “mature polypeptide codingsequence” is defined herein as a nucleotide sequence that encodes amature polypeptide having feruloyl esterase activity. In one aspect, themature polypeptide coding sequence is nucleotides 76 to 1821 of SEQ IDNO: 1 based on the SignalP program (Nielsen et al., 1997, supra) programthat predicts nucleotides 1 to 75 of SEQ ID NO: 1 encode a signalpeptide.

Sequence Identity: The relatedness between two amino acid sequences orbetween two nucleotide sequences is described by the parameter “sequenceidentity”.

For purposes of the present invention, the degree of sequence identitybetween two amino acid sequences is determined using theNeedleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol.48: 443-453) as implemented in the Needle program of the EMBOSS package(EMBOSS: The European Molecular Biology Open Software Suite, Rice etal., 2000, Trends in Genetics 16: 276-277), preferably version 3.0.0 orlater. The optional parameters used are gap open penalty of 10, gapextension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62)substitution matrix. The output of Needle labeled “longest identity”(obtained using the -nobrief option) is used as the percent identity andis calculated as follows:

(Identical Residues×100)/(Length of Alignment−Total Number of Gaps inAlignment)

For purposes of the present invention, the degree of sequence identitybetween two deoxyribonucleotide sequences is determined using theNeedleman-Wunsch algorithm (Needleman and Wunsch, 1970, supra) asimplemented in the Needle program of the EMBOSS package (EMBOSS: TheEuropean Molecular Biology Open Software Suite, Rice et al., 2000,supra), preferably version 3.0.0 or later. The optional parameters usedare gap open penalty of 10, gap extension penalty of 0.5, and theEDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix. The outputof Needle labeled “longest identity” (obtained using the -nobriefoption) is used as the percent identity and is calculated as follows:

(Identical Deoxyribonucleotides×100)/(Length of Alignment−Total Numberof Gaps in Alignment)

Homologous sequence: The term “homologous sequence” is defined herein asa predicted protein having an E value (or expectancy score) of less than0.001 in a tfasty search (Pearson, W. R., 1999, in BioinformaticsMethods and Protocols, S. Misener and S. A. Krawetz, ed., pp. 185-219)with the Thielavia terrestris feruloyl esterase of SEQ ID NO: 2 or themature polypeptide thereof.

Polypeptide fragment: The term “polypeptide fragment” is defined hereinas a polypeptide having one or more (several) amino acids deleted fromthe amino and/or carboxyl terminus of the mature polypeptide of SEQ IDNO: 2; or a homologous sequence thereof; wherein the fragment hasferuloyl esterase activity. In one aspect, a fragment contains at least485 amino acid residues, more preferably at least 510 amino acidresidues, and most preferably at least 535 amino acid residues, of themature polypeptide of SEQ ID NO: 2 or a homologous sequence thereof.

Subsequence: The term “subsequence” is defined herein as a nucleotidesequence having one or more (several) nucleotides deleted from the 5′and/or 3′ end of the mature polypeptide coding sequence of SEQ ID NO: 1;or a homologous sequence thereof; wherein the subsequence encodes apolypeptide fragment having feruloyl esterase activity. In one aspect, asubsequence contains at least 1455 nucleotides, more preferably at least1530 nucleotides, and most preferably at least 1605 nucleotides of themature polypeptide coding sequence of SEQ ID NO: 1 or a homologoussequence thereof.

Allelic variant: The term “allelic variant” denotes herein any of two ormore alternative forms of a gene occupying the same chromosomal locus.Allelic variation arises naturally through mutation, and may result inpolymorphism within populations. Gene mutations can be silent (no changein the encoded polypeptide) or may encode polypeptides having alteredamino acid sequences. An allelic variant of a polypeptide is apolypeptide encoded by an allelic variant of a gene.

Isolated polynucleotide: The term “isolated polynucleotide” as usedherein refers to a polynucleotide that is isolated from a source. In apreferred aspect, the polynucleotide is at least 1% pure, preferably atleast 5% pure, more preferably at least 10% pure, more preferably atleast 20% pure, more preferably at least 40% pure, more preferably atleast 60% pure, even more preferably at least 80% pure, and mostpreferably at least 90% pure, as determined by agarose electrophoresis.

Substantially pure polynucleotide: The term “substantially purepolynucleotide” as used herein refers to a polynucleotide preparationfree of other extraneous or unwanted nucleotides and in a form suitablefor use within genetically engineered protein production systems. Thus,a substantially pure polynucleotide contains at most 10%, preferably atmost 8%, more preferably at most 6%, more preferably at most 5%, morepreferably at most 4%, more preferably at most 3%, even more preferablyat most 2%, most preferably at most 1%, and even most preferably at most0.5% by weight of other polynucleotide material with which it isnatively or recombinantly associated. A substantially purepolynucleotide may, however, include naturally occurring 5′ and 3′untranslated regions, such as promoters and terminators. It is preferredthat the substantially pure polynucleotide is at least 90% pure,preferably at least 92% pure, more preferably at least 94% pure, morepreferably at least 95% pure, more preferably at least 96% pure, morepreferably at least 97% pure, even more preferably at least 98% pure,most preferably at least 99% pure, and even most preferably at least99.5% pure by weight. The polynucleotides of the present invention arepreferably in a substantially pure form, i.e., that the polynucleotidepreparation is essentially free of other polynucleotide material withwhich it is natively or recombinantly associated. The polynucleotidesmay be of genomic, cDNA, RNA, semisynthetic, synthetic origin, or anycombinations thereof.

Coding sequence: When used herein the term “coding sequence” means anucleotide sequence, which directly specifies the amino acid sequence ofits protein product. The boundaries of the coding sequence are generallydetermined by an open reading frame, which usually begins with the ATGstart codon or alternative start codons such as GTG and TTG and endswith a stop codon such as TAA, TAG, and TGA. The coding sequence may bea DNA, cDNA, synthetic, or recombinant nucleotide sequence.

cDNA: The term “cDNA” is defined herein as a DNA molecule that can beprepared by reverse transcription from a mature, spliced, mRNA moleculeobtained from a eukaryotic cell. cDNA lacks intron sequences that may bepresent in the corresponding genomic DNA. The initial, primary RNAtranscript is a precursor to mRNA that is processed through a series ofsteps before appearing as mature spliced mRNA. These steps include theremoval of intron sequences by a process called splicing. cDNA derivedfrom mRNA lacks, therefore, any intron sequences.

Nucleic acid construct: The term “nucleic acid construct” as used hereinrefers to a nucleic acid molecule, either single- or double-stranded,which is isolated from a naturally occurring gene or which is modifiedto contain segments of nucleic acids in a manner that would nototherwise exist in nature or which is synthetic. The term nucleic acidconstruct is synonymous with the term “expression cassette” when thenucleic acid construct contains the control sequences required forexpression of a coding sequence of the present invention.

Control sequences: The term “control sequences” is defined herein toinclude all components necessary for the expression of a polynucleotideencoding a polypeptide of the present invention. Each control sequencemay be native or foreign to the nucleotide sequence encoding thepolypeptide or native or foreign to each other. Such control sequencesinclude, but are not limited to, a leader, polyadenylation sequence,propeptide sequence, promoter, signal peptide sequence, andtranscription terminator. At a minimum, the control sequences include apromoter, and transcriptional and translational stop signals. Thecontrol sequences may be provided with linkers for the purpose ofintroducing specific restriction sites facilitating ligation of thecontrol sequences with the coding region of the nucleotide sequenceencoding a polypeptide.

Operably linked: The term “operably linked” denotes herein aconfiguration in which a control sequence is placed at an appropriateposition relative to the coding sequence of a polynucleotide sequencesuch that the control sequence directs the expression of the codingsequence of a polypeptide.

Expression: The term “expression” includes any step involved in theproduction of a polypeptide including, but not limited to,transcription, post-transcriptional modification, translation,post-translational modification, and secretion.

Expression vector: The term “expression vector” is defined herein as alinear or circular DNA molecule that comprises a polynucleotide encodinga polypeptide of the present invention and is operably linked toadditional nucleotides that provide for its expression.

Host cell: The term “host cell”, as used herein, includes any cell typethat is susceptible to transformation, transfection, transduction, andthe like with a nucleic acid construct or expression vector comprising apolynucleotide of the present invention.

Modification: The term “modification” means herein any chemicalmodification of the polypeptide comprising or consisting of the maturepolypeptide of SEQ ID NO: 2; or a homologous sequence thereof; as wellas genetic manipulation of the DNA encoding such a polypeptide. Themodification can be a substitution, a deletion and/or an insertion ofone or more (several) amino acids as well as replacements of one or more(several) amino acid side chains.

Artificial variant: When used herein, the term “artificial variant”means a polypeptide having feruloyl esterase activity produced by anorganism expressing a modified polynucleotide sequence of the maturepolypeptide coding sequence of SEQ ID NO: 1; or a homologous sequencethereof. The modified nucleotide sequence is obtained through humanintervention by modification of the polynucleotide sequence disclosed inSEQ ID NO: 1; or a homologous sequence thereof.

DETAILED DESCRIPTION OF THE INVENTION Polypeptides Having FeruloylEsterase Activity

In a first aspect, the present invention relates to isolatedpolypeptides comprising amino acid sequences having a degree of sequenceidentity to the mature polypeptide of SEQ ID NO: 2 of preferably atleast 75%, more preferably at least 80%, more preferably at least 85%,even more preferably at least 90%, most preferably at least 95%, andeven most preferably at least 96%, at least 97%, at least 98%, or atleast 99%, which have feruloyl esterase activity (hereinafter“homologous polypeptides”). In a preferred aspect, the homologouspolypeptides comprise amino acid sequences that differ by ten aminoacids, preferably by five amino acids, more preferably by four aminoacids, even more preferably by three amino acids, most preferably by twoamino acids, and even most preferably by one amino acid from the maturepolypeptide of SEQ ID NO: 2.

A polypeptide of the present invention preferably comprises the aminoacid sequence of SEQ ID NO: 2 or an allelic variant thereof; or afragment thereof having feruloyl esterase activity. In a preferredaspect, the polypeptide comprises the amino acid sequence of SEQ ID NO:2. In another preferred aspect, the polypeptide comprises the maturepolypeptide of SEQ ID NO: 2. In another preferred aspect, thepolypeptide comprises amino acids 26 to 585 of SEQ ID NO: 2, or anallelic variant thereof; or a fragment thereof having feruloyl esteraseactivity. In another preferred aspect, the polypeptide comprises aminoacids 26 to 585 of SEQ ID NO: 2. In another preferred aspect, thepolypeptide consists of the amino acid sequence of SEQ ID NO: 2 or anallelic variant thereof; or a fragment thereof having feruloyl esteraseactivity. In another preferred aspect, the polypeptide consists of theamino acid sequence of SEQ ID NO: 2. In another preferred aspect, thepolypeptide consists of the mature polypeptide of SEQ ID NO: 2. Inanother preferred aspect, the polypeptide consists of amino acids 26 to585 of SEQ ID NO: 2 or an allelic variant thereof; or a fragment thereofhaving feruloyl esterase activity. In another preferred aspect, thepolypeptide consists of amino acids 26 to 585 of SEQ ID NO: 2.

In a second aspect, the present invention relates to isolatedpolypeptides having feruloyl esterase activity that are encoded bypolynucleotides that hybridize under preferably very low stringencyconditions, more preferably low stringency conditions, more preferablymedium stringency conditions, more preferably medium-high stringencyconditions, even more preferably high stringency conditions, and mostpreferably very high stringency conditions with (i) the maturepolypeptide coding sequence of SEQ ID NO: 1, (ii) the cDNA sequencecontained in the mature polypeptide coding sequence of SEQ ID NO: 1, or(iii) a full-length complementary strand of (i) or (ii) (J. Sambrook, E.F. Fritsch, and T. Maniatis, 1989, Molecular Cloning, A LaboratoryManual, 2d edition, Cold Spring Harbor, N.Y.).

The nucleotide sequence of SEQ ID NO: 1; or a subsequence thereof; aswell as the amino acid sequence of SEQ ID NO: 2; or a fragment thereof;may be used to design nucleic acid probes to identify and clone DNAencoding polypeptides having feruloyl esterase activity from strains ofdifferent genera or species according to methods well known in the art.In particular, such probes can be used for hybridization with thegenomic or cDNA of the genus or species of interest, following standardSouthern blotting procedures, in order to identify and isolate thecorresponding gene therein. Such probes can be considerably shorter thanthe entire sequence, but should be at least 14, preferably at least 25,more preferably at least 35, and most preferably at least 70 nucleotidesin length. It is, however, preferred that the nucleic acid probe is atleast 100 nucleotides in length. For example, the nucleic acid probe maybe at least 200 nucleotides, preferably at least 300 nucleotides, morepreferably at least 400 nucleotides, or most preferably at least 500nucleotides in length. Even longer probes may be used, e.g., nucleicacid probes that are preferably at least 600 nucleotides, morepreferably at least 700 nucleotides, even more preferably at least 800nucleotides, or most preferably at least 900 nucleotides in length. BothDNA and RNA probes can be used. The probes are typically labeled fordetecting the corresponding gene (for example, with ³²P, ³H, ³⁵S,biotin, or avidin). Such probes are encompassed by the presentinvention.

A genomic DNA or cDNA library prepared from such other strains may,therefore, be screened for DNA that hybridizes with the probes describedabove and encodes a polypeptide having feruloyl esterase activity.Genomic or other DNA from such other strains may be separated by agaroseor polyacrylamide gel electrophoresis, or other separation techniques.DNA from the libraries or the separated DNA may be transferred to andimmobilized on nitrocellulose or other suitable carrier material. Inorder to identify a clone or DNA that is homologous with SEQ ID NO: 1,or a subsequence thereof, the carrier material is preferably used in aSouthern blot.

For purposes of the present invention, hybridization indicates that thenucleotide sequence hybridizes to a labeled nucleic acid probecorresponding to the mature polypeptide coding sequence of SEQ ID NO: 1;the cDNA sequence contained in the mature polypeptide coding sequence ofSEQ ID NO: 1; its full-length complementary strand; or a subsequencethereof; under very low to very high stringency conditions. Molecules towhich the nucleic acid probe hybridizes under these conditions can bedetected using, for example, X-ray film.

In a preferred aspect, the nucleic acid probe is the mature polypeptidecoding sequence of SEQ ID NO: 1. In another preferred aspect, thenucleic acid probe is nucleotides 76 to 1821 of SEQ ID NO: 1. In anotherpreferred aspect, the nucleic acid probe is a polynucleotide sequencethat encodes the polypeptide of SEQ ID NO: 2, or a subsequence thereof.In another preferred aspect, the nucleic acid probe is SEQ ID NO: 1. Inanother preferred aspect, the nucleic acid probe is the polynucleotidesequence contained in plasmid pDFng105 which is contained in E. coliNRRL B-50204, wherein the polynucleotide sequence thereof encodes apolypeptide having feruloyl esterase activity. In another preferredaspect, the nucleic acid probe is the mature polypeptide coding regioncontained in plasmid pDFng105 which is contained in E. coli NRRLB-50204.

For long probes of at least 100 nucleotides in length, very low to veryhigh stringency conditions are defined as prehybridization andhybridization at 42° C. in 5×SSPE, 0.3% SDS, 200 μg/ml sheared anddenatured salmon sperm DNA, and either 25% formamide for very low andlow stringencies, 35% formamide for medium and medium-high stringencies,or 50% formamide for high and very high stringencies, following standardSouthern blotting procedures for 12 to 24 hours optimally.

For long probes of at least 100 nucleotides in length, the carriermaterial is finally washed three times each for 15 minutes using 2×SSC,0.2% SDS preferably at 45° C. (very low stringency), more preferably at50° C. (low stringency), more preferably at 55° C. (medium stringency),more preferably at 60° C. (medium-high stringency), even more preferablyat 65° C. (high stringency), and most preferably at 70° C. (very highstringency).

For short probes of about 15 nucleotides to about 70 nucleotides inlength, stringency conditions are defined as prehybridization,hybridization, and washing post-hybridization at about 5° C. to about10° C. below the calculated T_(m) using the calculation according toBolton and McCarthy (1962, Proceedings of the National Academy ofSciences USA 48:1390) in 0.9 M NaCl, 0.09 M Tris-HCl pH 7.6, 6 mM EDTA,0.5% NP-40, 1×Denhardt's solution, 1 mM sodium pyrophosphate, 1 mMsodium monobasic phosphate, 0.1 mM ATP, and 0.2 mg of yeast RNA per mlfollowing standard Southern blotting procedures for 12 to 24 hoursoptimally.

For short probes of about 15 nucleotides to about 70 nucleotides inlength, the carrier material is washed once in 6×SCC plus 0.1% SDS for15 minutes and twice each for 15 minutes using 6×SSC at 5° C. to 10° C.below the calculated T_(m).

In a third aspect, the present invention relates to isolatedpolypeptides having feruloyl esterase activity encoded bypolynucleotides comprising or consisting of nucleotide sequences havinga degree of sequence identity to the mature polypeptide coding sequenceof SEQ ID NO: 1 of preferably at least 75%, more preferably at least80%, more preferably at least 85%, even more preferably at least 90%,most preferably at least 95%, and even most preferably at least 96%, atleast 97%, at least 98%, or at least 99%, which encode a polypeptidehaving feruloyl esterase activity. See polynucleotide section herein.

In a fourth aspect, the present invention relates to artificial variantscomprising a substitution, deletion, and/or insertion of one or more (orseveral) amino acids of the mature polypeptide of SEQ ID NO: 2, or ahomologous sequence thereof. Preferably, amino acid changes are of aminor nature, that is conservative amino acid substitutions orinsertions that do not significantly affect the folding and/or activityof the protein; small deletions, typically of one to about 30 aminoacids; small amino- or carboxyl-terminal extensions, such as anamino-terminal methionine residue; a small linker peptide of up to about20-25 residues; or a small extension that facilitates purification bychanging net charge or another function, such as a poly-histidine tract,an antigenic epitope or a binding domain.

Examples of conservative substitutions are within the group of basicamino acids (arginine, lysine and histidine), acidic amino acids(glutamic acid and aspartic acid), polar amino acids (glutamine andasparagine), hydrophobic amino acids (leucine, isoleucine and valine),aromatic amino acids (phenylalanine, tryptophan and tyrosine), and smallamino acids (glycine, alanine, serine, threonine and methionine). Aminoacid substitutions that do not generally alter specific activity areknown in the art and are described, for example, by H. Neurath and R. L.Hill, 1979, In, The Proteins, Academic Press, New York. The mostcommonly occurring exchanges are Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser,Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Tyr/Phe, Ala/Pro, Lys/Arg,Asp/Asn, Leu/Ile, Leu/Val, Ala/Glu, and Asp/Gly.

In addition to the 20 standard amino acids, non-standard amino acids(such as 4-hydroxyproline, 6-N-methyl lysine, 2-aminoisobutyric acid,isovaline, and alpha-methyl serine) may be substituted for amino acidresidues of a wild-type polypeptide. A limited number ofnon-conservative amino acids, amino acids that are not encoded by thegenetic code, and unnatural amino acids may be substituted for aminoacid residues. “Unnatural amino acids” have been modified after proteinsynthesis, and/or have a chemical structure in their side chain(s)different from that of the standard amino acids. Unnatural amino acidscan be chemically synthesized, and preferably, are commerciallyavailable, and include pipecolic acid, thiazolidine carboxylic acid,dehydroproline, 3- and 4-methylproline, and 3,3-dimethylproline.

Alternatively, the amino acid changes are of such a nature that thephysico-chemical properties of the polypeptides are altered. Forexample, amino acid changes may improve the thermal stability of thepolypeptide, alter the substrate specificity, change the pH optimum, andthe like.

Essential amino acids in the parent polypeptide can be identifiedaccording to procedures known in the art, such as site-directedmutagenesis or alanine-scanning mutagenesis (Cunningham and Wells, 1989,Science 244: 1081-1085). In the latter technique, single alaninemutations are introduced at every residue in the molecule, and theresultant mutant molecules are tested for biological activity (i.e.,feruloyl esterase activity) to identify amino acid residues that arecritical to the activity of the molecule. See also, Hilton et al., 1996,J. Biol. Chem. 271: 4699-4708. The active site of the enzyme or otherbiological interaction can also be determined by physical analysis ofstructure, as determined by such techniques as nuclear magneticresonance, crystallography, electron diffraction, or photoaffinitylabeling, in conjunction with mutation of putative contact site aminoacids. See, for example, de Vos et al., 1992, Science 255: 306-312;Smith et al., 1992, J. Mol. Biol. 224: 899-904; Wlodaver et al., 1992,FEBS Lett. 309: 59-64. The identities of essential amino acids can alsobe inferred from analysis of identities with polypeptides that arerelated to a polypeptide according to the invention.

Single or multiple amino acid substitutions, deletions, and/orinsertions can be made and tested using known methods of mutagenesis,recombination, and/or shuffling, followed by a relevant screeningprocedure, such as those disclosed by Reidhaar-Olson and Sauer, 1988,Science 241: 53-57; Bowie and Sauer, 1989, Proc. Natl. Acad. Sci. USA86: 2152-2156; WO 95/17413; or WO 95/22625. Other methods that can beused include error-prone PCR, phage display (e.g., Lowman et al., 1991,Biochem. 30: 10832-10837; U.S. Pat. No. 5,223,409; WO 92/06204), andregion-directed mutagenesis (Derbyshire et al., 1986, Gene 46: 145; Neret al., 1988, DNA 7: 127).

Mutagenesis/shuffling methods can be combined with high-throughput,automated screening methods to detect activity of cloned, mutagenizedpolypeptides expressed by host cells (Ness et al., 1999, NatureBiotechnology 17: 893-896). Mutagenized DNA molecules that encode activepolypeptides can be recovered from the host cells and rapidly sequencedusing standard methods in the art. These methods allow the rapiddetermination of the importance of individual amino acid residues in apolypeptide of interest, and can be applied to polypeptides of unknownstructure.

The total number of amino acid substitutions, deletions and/orinsertions of the mature polypeptide of SEQ ID NO: 2 is 10, preferably9, more preferably 8, more preferably 7, more preferably at most 6, morepreferably 5, more preferably 4, even more preferably 3, most preferably2, and even most preferably 1.

Sources of Polypeptides Having Feruloyl Esterase Activity

A polypeptide having feruloyl esterase activity of the present inventionmay be obtained from microorganisms of any genus. For purposes of thepresent invention, the term “obtained from” as used herein in connectionwith a given source shall mean that the polypeptide encoded by anucleotide sequence is produced by the source or by a strain in whichthe nucleotide sequence from the source has been inserted. In apreferred aspect, the polypeptide obtained from a given source issecreted extracellularly.

A polypeptide having feruloyl esterase activity of the present inventionmay be a bacterial polypeptide. For example, the polypeptide may be agram positive bacterial polypeptide such as a Bacillus, Streptococcus,Streptomyces, Staphylococcus, Enterococcus, Lactobacillus, Lactococcus,Clostridium, Geobacillus, or Oceanobacillus polypeptide having feruloylesterase activity, or a Gram negative bacterial polypeptide such as anE. coli, Pseudomonas, Salmonella, Campylobacter, Helicobacter,Flavobacterium, Fusobacterium, Ilyobacter, Neisseria, or Ureaplasmapolypeptide having feruloyl esterase activity.

In a preferred aspect, the polypeptide is a Bacillus alkalophilus,Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans,Bacillus clausii, Bacillus coagulans, Bacillus firmus, Bacillus lautus,Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacilluspumilus, Bacillus stearothermophilus, Bacillus subtilis, or Bacillusthuringiensis polypeptide having feruloyl esterase activity.

In another preferred aspect, the polypeptide is a Streptococcusequisimilis, Streptococcus pyogenes, Streptococcus uberis, orStreptococcus equi subsp. Zooepidemicus polypeptide having feruloylesterase activity.

In another preferred aspect, the polypeptide is a Streptomycesachromogenes, Streptomyces avermitilis, Streptomyces coelicolor,Streptomyces griseus, or Streptomyces lividans polypeptide havingferuloyl esterase activity.

A polypeptide having feruloyl esterase activity of the present inventionmay also be a fungal polypeptide, and more preferably a yeastpolypeptide such as a Candida, Kluyveromyces, Pichia, Saccharomyces,Schizosaccharomyces, or Yarrowia polypeptide having feruloyl esteraseactivity; or more preferably a filamentous fungal polypeptide such as anAcremonium, Agaricus, Alternaria, Aspergillus, Aureobasidium,Botryospaeria, Ceriporiopsis, Chaetomidium, Chrysosporium, Claviceps,Cochliobolus, Coprinopsis, Coptotermes, Corynascus, Cryphonectria,Cryptococcus, Diplodia, Exidia, Filibasidium, Fusarium, Gibberella,Holomastigotoides, Humicola, Irpex, Lentinula, Leptospaeria,Magnaporthe, Melanocarpus, Meripilus, Mucor, Myceliophthora,Neocallimastix, Neurospora, Paecilomyces, Penicillium, Phanerochaete,Piromyces, Poitrasia, Pseudoplectania, Pseudotrichonympha, Rhizomucor,Schizophyllum, Scytalidium, Talaromyces, Thermoascus, Thielavia,Tolypocladium, Trichoderma, Trichophaea, Verticillium, Volvariella, orXylaria polypeptide having feruloyl esterase activity.

In a preferred aspect, the polypeptide is a Saccharomycescarlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus,Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomycesnorbensis, or Saccharomyces oviformis polypeptide having feruloylesterase activity.

In another preferred aspect, the polypeptide is an Acremoniumcellulolyticus, Aspergillus aculeatus, Aspergillus awamori, Aspergillusfumigatus, Aspergillus foetidus, Aspergillus japonicus, Aspergillusnidulans, Aspergillus niger, Aspergillus oryzae, Chrysosporiumkeratinophilum, Chrysosporium lucknowense, Chrysosporium tropicum,Chrysosporium merdarium, Chrysosporium inops, Chrysosporium pannicola,Chrysosporium queenslandicum, Chrysosporium zonatum, Fusariumbactridioides, Fusarium cerealis, Fusarium crookwellense, Fusariumculmorum, Fusarium graminearum, Fusarium graminum, Fusariumheterosporum, Fusarium negundi, Fusarium oxysporum, Fusariumreticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum,Fusarium sporotrichioides, Fusarium sulphureum, Fusarium torulosum,Fusarium trichothecioides, Fusarium venenatum, Humicola grisea, Humicolainsolens, or Humicola lanuginosa, Irpex lacteus, Mucor miehei,Myceliophthora thermophila, Neurospora crassa, Penicillium funiculosum,Penicillium purpurogenum, Phanerochaete chrysosporium, Thielaviaachromatica, Thielavia albomyces, Thielavia albopilosa, Thielaviaaustraleinsis, Thielavia fimeti, Thielavia microspora, Thielaviaovispora, Thielavia peruviana, Thielavia spededonium, Thielavia setosa,Thielavia subthermophila, Thielavia terrestris, Trichoderma harzianum,Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei,or Trichoderma viride polypeptide having feruloyl esterase activity.

In another preferred aspect, the polypeptide is a Thielavia achromatica,Thielavia albomyces, Thielavia albopilosa, Thielavia australeinsis,Thielavia fimeti, Thielavia microspora, Thielavia ovispora, Thielaviaperuviana, Thielavia spededonium, Thielavia setosa, Thielaviasubthermophila, or Thielavia terrestris polypeptide.

In a more preferred aspect, the polypeptide is a Thielavia terrestrispolypeptide having feruloyl esterase activity. In a most preferredaspect, the polypeptide is a Thielavia terrestris NRRL 8126 polypeptidehaving feruloyl esterase activity, e.g., the polypeptide comprising themature polypeptide of SEQ ID NO: 2.

It will be understood that for the aforementioned species the inventionencompasses both the perfect and imperfect states, and other taxonomicequivalents, e.g., anamorphs, regardless of the species name by whichthey are known. Those skilled in the art will readily recognize theidentity of appropriate equivalents.

Strains of these species are readily accessible to the public in anumber of culture collections, such as the American Type CultureCollection (ATCC), Deutsche Sammlung von Mikroorganismen andZellkulturen GmbH (DSM), Centraalbureau Voor Schimmelcultures (CBS), andAgricultural Research Service Patent Culture Collection, NorthernRegional Research Center (NRRL).

Furthermore, such polypeptides may be identified and obtained from othersources including microorganisms isolated from nature (e.g., soil,composts, water, etc.) using the above-mentioned probes. Techniques forisolating microorganisms from natural habitats are well known in theart. The polynucleotide may then be obtained by similarly screening agenomic or cDNA library of such a microorganism. Once a polynucleotideencoding a polypeptide has been detected with the probe(s), thepolynucleotide can be isolated or cloned by utilizing techniques thatare well known to those of ordinary skill in the art (see, e.g.,Sambrook et al., 1989, supra).

Polypeptides of the present invention also include fused polypeptides orcleavable fusion polypeptides in which another polypeptide is fused atthe N-terminus or the C-terminus of the polypeptide or fragment thereof.A fused polypeptide is produced by fusing a nucleotide sequence (or aportion thereof) encoding another polypeptide to a nucleotide sequence(or a portion thereof) of the present invention. Techniques forproducing fusion polypeptides are known in the art, and include ligatingthe coding sequences encoding the polypeptides so that they are in frameand that expression of the fused polypeptide is under control of thesame promoter(s) and terminator.

A fusion polypeptide can further comprise a cleavage site. Uponsecretion of the fusion protein, the site is cleaved releasing thepolypeptide having feruloyl esterase activity from the fusion protein.Examples of cleavage sites include, but are not limited to, a Kex2 sitethat encodes the dipeptide Lys-Arg (Martin et al., 2003, J. Ind.Microbiol. Biotechnol. 3: 568-576; Svetina et al., 2000, J. Biotechnol.76: 245-251; Rasmussen-Wilson et al., 1997, Appl. Environ. Microbiol.63: 3488-3493; Ward et al., 1995, Biotechnology 13: 498-503; andContreras et al., 1991, Biotechnology 9: 378-381), an Ile-(Glu orAsp)-Gly-Arg site, which is cleaved by a Factor Xa protease after thearginine residue (Eaton et al., 1986, Biochem. 25: 505-512); aAsp-Asp-Asp-Asp-Lys site, which is cleaved by an enterokinase after thelysine (Collins-Racie et al., 1995, Biotechnology 13: 982-987); aHis-Tyr-Glu site or His-Tyr-Asp site, which is cleaved by Genenase I(Carter et al., 1989, Proteins: Structure, Function, and Genetics 6:240-248); a Leu-Val-Pro-Arg-Gly-Ser site, which is cleaved by thrombinafter the Arg (Stevens, 2003, Drug Discovery World 4: 35-48); aGlu-Asn-Leu-Tyr-Phe-Gln-Gly site, which is cleaved by TEV protease afterthe Gln (Stevens, 2003, supra); and a Leu-Glu-Val-Leu-Phe-Gln-Gly-Prosite, which is cleaved by a genetically engineered form of humanrhinovirus 3C protease after the Gln (Stevens, 2003, supra).

Polynucleotides

The present invention also relates to isolated polynucleotidescomprising or consisting of nucleotide sequences that encodepolypeptides having feruloyl esterase activity of the present invention.

In a preferred aspect, the nucleotide sequence comprises or consists ofSEQ ID NO: 1. In another more preferred aspect, the nucleotide sequencecomprises or consists of the sequence contained in plasmid pDFng105which is contained in E. coli NRRL B-50204. In another preferred aspect,the nucleotide sequence comprises or consists of the mature polypeptidecoding sequence of SEQ ID NO: 1. In another preferred aspect, thenucleotide sequence comprises or consists of nucleotides 76 to 1821 ofSEQ ID NO: 1. In another more preferred aspect, the nucleotide sequencecomprises or consists of the mature polypeptide coding sequencecontained in plasmid pDFng105 which is contained in E. coli NRRLB-50204. The present invention also encompasses nucleotide sequencesthat encode polypeptides comprising or consisting of the amino acidsequence of SEQ ID NO: 2 or the mature polypeptide thereof, which differfrom SEQ ID NO: 1 or the mature polypeptide coding sequence thereof byvirtue of the degeneracy of the genetic code. The present invention alsorelates to subsequences of SEQ ID NO: 1 that encode fragments of SEQ IDNO: 2 having feruloyl esterase activity.

The present invention also relates to mutant polynucleotides comprisingor consisting of at least one mutation in the mature polypeptide codingsequence of SEQ ID NO: 1, in which the mutant nucleotide sequenceencodes the mature polypeptide of SEQ ID NO: 2.

The techniques used to isolate or clone a polynucleotide encoding apolypeptide are known in the art and include isolation from genomic DNA,preparation from cDNA, or a combination thereof. The cloning of thepolynucleotides of the present invention from such genomic DNA can beeffected, e.g., by using the well known polymerase chain reaction (PCR)or antibody screening of expression libraries to detect cloned DNAfragments with shared structural features. See, e.g., Innis et al.,1990, PCR: A Guide to Methods and Application, Academic Press, New York.Other nucleic acid amplification procedures such as ligase chainreaction (LCR), ligated activated transcription (LAT) and nucleotidesequence-based amplification (NASBA) may be used. The polynucleotidesmay be cloned from a strain of Thielavia, or another or related organismand thus, for example, may be an allelic or species variant of thepolypeptide encoding region of the nucleotide sequence.

The present invention also relates to isolated polynucleotidescomprising or consisting of nucleotide sequences having a degree ofsequence identity to the mature polypeptide coding sequence of SEQ IDNO: 1 of preferably at least 75%, more preferably at least 80%, morepreferably at least 85%, even more preferably at least 90%, mostpreferably at least 95%, and even most preferably at least 96%, at least97%, at least 98%, or at least 99%, which encode a polypeptide havingferuloyl esterase activity.

Modification of a nucleotide sequence encoding a polypeptide of thepresent invention may be necessary for the synthesis of polypeptidessubstantially similar to the polypeptide. The term “substantiallysimilar” to the polypeptide refers to non-naturally occurring forms ofthe polypeptide. These polypeptides may differ in some engineered wayfrom the polypeptide isolated from its native source, e.g., artificialvariants that differ in specific activity, thermostability, pH optimum,or the like. The variant sequence may be constructed on the basis of thenucleotide sequence presented as the mature polypeptide coding sequenceof SEQ ID NO: 1, e.g., a subsequence thereof, and/or by introduction ofnucleotide substitutions that do not give rise to another amino acidsequence of the polypeptide encoded by the nucleotide sequence, butwhich correspond to the codon usage of the host organism intended forproduction of the enzyme, or by introduction of nucleotide substitutionsthat may give rise to a different amino acid sequence. For a generaldescription of nucleotide substitution, see, e.g., Ford et al., 1991,Protein Expression and Purification 2: 95-107.

It will be apparent to those skilled in the art that such substitutionscan be made outside the regions critical to the function of the moleculeand still result in an active polypeptide. Amino acid residues essentialto the activity of the polypeptide encoded by an isolated polynucleotideof the invention, and therefore preferably not subject to substitution,may be identified according to procedures known in the art, such assite-directed mutagenesis or alanine-scanning mutagenesis (see, e.g.,Cunningham and Wells, 1989, supra). In the latter technique, mutationsare introduced at every positively charged residue in the molecule, andthe resultant mutant molecules are tested for feruloyl esterase activityto identify amino acid residues that are critical to the activity of themolecule. Sites of substrate-enzyme interaction can also be determinedby analysis of the three-dimensional structure as determined by suchtechniques as nuclear magnetic resonance analysis, crystallography orphotoaffinity labeling (see, e.g., de Vos et al., 1992, supra; Smith etal., 1992, supra; Wlodaver et al., 1992, supra).

The present invention also relates to isolated polynucleotides encodingpolypeptides of the present invention, which hybridize under preferablyvery low stringency conditions, more preferably low stringencyconditions, more preferably medium stringency conditions, morepreferably medium-high stringency conditions, even more preferably highstringency conditions, and most preferably very high stringencyconditions with (i) the mature polypeptide coding sequence of SEQ ID NO:1, (ii) the cDNA sequence contained in the mature polypeptide codingsequence of SEQ ID NO: 1, or (iii) a full-length complementary strand of(i) or (ii); or allelic variants and subsequences thereof (Sambrook etal., 1989, supra), as defined herein.

The present invention also relates to isolated polynucleotides obtainedby (a) hybridizing a population of DNA under preferably very lowstringency conditions, more preferably low stringency conditions, morepreferably medium stringency conditions, more preferably medium-highstringency conditions, even more preferably high stringency conditions,and most preferably very high stringency conditions with (i) the maturepolypeptide coding sequence of SEQ ID NO: 1, (ii) the cDNA sequencecontained in the mature polypeptide coding sequence of SEQ ID NO: 1, or(iii) a full-length complementary strand of (i) or (ii); and (b)isolating the hybridizing polynucleotide, which encodes a polypeptidehaving feruloyl esterase activity.

Nucleic Acid Constructs

The present invention also relates to nucleic acid constructs comprisingan isolated polynucleotide of the present invention operably linked toone or more (several) control sequences that direct the expression ofthe coding sequence in a suitable host cell under conditions compatiblewith the control sequences.

An isolated polynucleotide encoding a polypeptide of the presentinvention may be manipulated in a variety of ways to provide forexpression of the polypeptide. Manipulation of the polynucleotide'ssequence prior to its insertion into a vector may be desirable ornecessary depending on the expression vector. The techniques formodifying polynucleotide sequences utilizing recombinant DNA methods arewell known in the art.

The control sequence may be an appropriate promoter sequence, anucleotide sequence that is recognized by a host cell for expression ofa polynucleotide encoding a polypeptide of the present invention. Thepromoter sequence contains transcriptional control sequences thatmediate the expression of the polypeptide. The promoter may be anynucleotide sequence that shows transcriptional activity in the host cellof choice including mutant, truncated, and hybrid promoters, and may beobtained from genes encoding extracellular or intracellular polypeptideseither homologous or heterologous to the host cell.

Examples of suitable promoters for directing the transcription of thenucleic acid constructs of the present invention, especially in abacterial host cell, are the promoters obtained from the E. coli lacoperon, Streptomyces coelicolor agarase gene (dagA), Bacillus subtilislevansucrase gene (sacB), Bacillus licheniformis alpha-amylase gene(amyL), Bacillus stearothermophilus maltogenic amylase gene (amyM),Bacillus amyloliquefaciens alpha-amylase gene (amyQ), Bacilluslicheniformis penicillinase gene (penP), Bacillus subtilis xylA and xylBgenes, and prokaryotic beta-lactamase gene (Villa-Kamaroff et al., 1978,Proceedings of the National Academy of Sciences USA 75: 3727-3731), aswell as the tac promoter (DeBoer et al., 1983, Proceedings of theNational Academy of Sciences USA 80: 21-25). Further promoters aredescribed in “Useful proteins from recombinant bacteria” in ScientificAmerican, 1980, 242: 74-94; and in Sambrook et al., 1989, supra.

Examples of suitable promoters for directing the transcription of thenucleic acid constructs of the present invention in a filamentous fungalhost cell are promoters obtained from the genes for Aspergillus oryzaeTAKA amylase, Rhizomucor miehei aspartic proteinase, Aspergillus nigerneutral alpha-amylase, Aspergillus niger acid stable alpha-amylase,Aspergillus niger or Aspergillus awamori glucoamylase (glaA), Rhizomucormiehei lipase, Aspergillus oryzae alkaline protease, Aspergillus oryzaetriose phosphate isomerase, Aspergillus nidulans acetamidase, Fusariumvenenatum amyloglucosidase (WO 00/56900), Fusarium venenatum Daria (WO00/56900), Fusarium venenatum Quinn (WO 00/56900), Fusarium oxysporumtrypsin-like protease (WO 96/00787), Trichoderma reeseibeta-glucosidase, Trichoderma reesei cellobiohydrolase I, Trichodermareesei cellobiohydrolase II, Trichoderma reesei endoglucanase I,Trichoderma reesei endoglucanase II, Trichoderma reesei endoglucanaseIII, Trichoderma reesei endoglucanase IV, Trichoderma reeseiendoglucanase V, Trichoderma reesei xylanase I, Trichoderma reeseixylanase II, Trichoderma reesei beta-xylosidase, as well as a NA2-tpipromoter (a modified promoter including a gene encoding a neutralalpha-amylase in Aspergilli in which the untranslated leader has beenreplaced by an untranslated leader from a gene encoding triose phosphateisomerase in Aspergilli; non-limiting examples include modifiedpromoters including the gene encoding neutral alpha-amylase inAspergillus niger in which the untranslated leader has been replaced byan untranslated leader from the gene encoding triose phosphate isomerasein Aspergillus nidulans or Aspergillus oryzae); and mutant, truncated,and hybrid promoters thereof.

In a yeast host, useful promoters are obtained from the genes forSaccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiaegalactokinase (GAL1), Saccharomyces cerevisiae alcoholdehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH1, ADH2/GAP),Saccharomyces cerevisiae triose phosphate isomerase (TPI), Saccharomycescerevisiae metallothionein (CUP1), and Saccharomyces cerevisiae3-phosphoglycerate kinase. Other useful promoters for yeast host cellsare described by Romanos et al., 1992, Yeast 8: 423-488.

The control sequence may also be a suitable transcription terminatorsequence, a sequence recognized by a host cell to terminatetranscription. The terminator sequence is operably linked to the 3′terminus of the nucleotide sequence encoding the polypeptide. Anyterminator that is functional in the host cell of choice may be used inthe present invention.

Preferred terminators for filamentous fungal host cells are obtainedfrom the genes for Aspergillus oryzae TAKA amylase, Aspergillus nigerglucoamylase, Aspergillus nidulans anthranilate synthase, Aspergillusniger alpha-glucosidase, and Fusarium oxysporum trypsin-like protease.

Preferred terminators for yeast host cells are obtained from the genesfor Saccharomyces cerevisiae enolase, Saccharomyces cerevisiaecytochrome C (CYC1), and Saccharomyces cerevisiaeglyceraldehyde-3-phosphate dehydrogenase. Other useful terminators foryeast host cells are described by Romanos et al., 1992, supra.

The control sequence may also be a suitable leader sequence, anontranslated region of an mRNA that is important for translation by thehost cell. The leader sequence is operably linked to the 5′ terminus ofthe nucleotide sequence encoding the polypeptide. Any leader sequencethat is functional in the host cell of choice may be used in the presentinvention.

Preferred leaders for filamentous fungal host cells are obtained fromthe genes for Aspergillus oryzae TAKA amylase and Aspergillus nidulanstriose phosphate isomerase.

Suitable leaders for yeast host cells are obtained from the genes forSaccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae3-phosphoglycerate kinase, Saccharomyces cerevisiae alpha-factor, andSaccharomyces cerevisiae alcoholdehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH2/GAP).

The control sequence may also be a polyadenylation sequence, a sequenceoperably linked to the 3′ terminus of the nucleotide sequence and, whentranscribed, is recognized by the host cell as a signal to addpolyadenosine residues to transcribed mRNA. Any polyadenylation sequencethat is functional in the host cell of choice may be used in the presentinvention.

Preferred polyadenylation sequences for filamentous fungal host cellsare obtained from the genes for Aspergillus oryzae TAKA amylase,Aspergillus niger glucoamylase, Aspergillus nidulans anthranilatesynthase, Fusarium oxysporum trypsin-like protease, and Aspergillusniger alpha-glucosidase.

Useful polyadenylation sequences for yeast host cells are described byGuo and Sherman, 1995, Molecular Cellular Biology 15: 5983-5990.

The control sequence may also be a signal peptide coding sequence thatencodes a signal peptide linked to the amino terminus of a polypeptideand directs the encoded polypeptide into the cell's secretory pathway.The 5′ end of the coding sequence of the nucleotide sequence mayinherently contain a signal peptide coding sequence naturally linked intranslation reading frame with the segment of the coding sequence thatencodes the secreted polypeptide. Alternatively, the 5′ end of thecoding sequence may contain a signal peptide coding sequence that isforeign to the coding sequence. The foreign signal peptide codingsequence may be required where the coding sequence does not naturallycontain a signal peptide coding sequence. Alternatively, the foreignsignal peptide coding sequence may simply replace the natural signalpeptide coding sequence in order to enhance secretion of thepolypeptide. However, any signal peptide coding sequence that directsthe expressed polypeptide into the secretory pathway of a host cell ofchoice, i.e., secreted into a culture medium, may be used in the presentinvention.

Effective signal peptide coding sequences for bacterial host cells arethe signal peptide coding sequences obtained from the genes for BacillusNCIB 11837 maltogenic amylase, Bacillus stearothermophilusalpha-amylase, Bacillus licheniformis subtilisin, Bacillus licheniformisbeta-lactamase, Bacillus stearothermophilus neutral proteases (nprT,nprS, nprM), and Bacillus subtilis prsA. Further signal peptides aredescribed by Simonen and Palva, 1993, Microbiological Reviews 57:109-137.

Effective signal peptide coding sequences for filamentous fungal hostcells are the signal peptide coding sequences obtained from the genesfor Aspergillus oryzae TAKA amylase, Aspergillus niger neutral amylase,Aspergillus niger glucoamylase, Rhizomucor miehei aspartic proteinase,Humicola insolens cellulase, Humicola insolens endoglucanase V, andHumicola lanuginosa lipase.

Useful signal peptides for yeast host cells are obtained from the genesfor Saccharomyces cerevisiae alpha-factor and Saccharomyces cerevisiaeinvertase. Other useful signal peptide coding sequences are described byRomanos et al., 1992, supra.

In a preferred aspect, the signal peptide comprises or consists of aminoacids 1 to 25 of SEQ ID NO: 2. In another preferred aspect, the signalpeptide coding sequence comprises or consists of nucleotides 1 to 75 ofSEQ ID NO: 1.

The control sequence may also be a propeptide coding sequence thatencodes a propeptide positioned at the amino terminus of a polypeptide.The resultant polypeptide is known as a proenzyme or propolypeptide (ora zymogen in some cases). A propeptide is generally inactive and can beconverted to a mature active polypeptide by catalytic or autocatalyticcleavage of the propeptide from the propolypeptide. The propeptidecoding sequence may be obtained from the genes for Bacillus subtilisalkaline protease (aprE), Bacillus subtilis neutral protease (nprT),Saccharomyces cerevisiae alpha-factor, Rhizomucor miehei asparticproteinase, and Myceliophthora thermophila laccase (WO 95/33836).

Where both signal peptide and propeptide sequences are present at theamino terminus of a polypeptide, the propeptide sequence is positionednext to the amino terminus of a polypeptide and the signal peptidesequence is positioned next to the amino terminus of the propeptidesequence.

It may also be desirable to add regulatory sequences that allow theregulation of the expression of the polypeptide relative to the growthof the host cell. Examples of regulatory systems are those that causethe expression of the gene to be turned on or off in response to achemical or physical stimulus, including the presence of a regulatorycompound. Regulatory systems in prokaryotic systems include the lac,tac, and trp operator systems. In yeast, the ADH2 system or GAL1 systemmay be used. In filamentous fungi, the TAKA alpha-amylase promoter,Aspergillus niger glucoamylase promoter, and Aspergillus oryzaeglucoamylase promoter may be used as regulatory sequences. Otherexamples of regulatory sequences are those that allow for geneamplification. In eukaryotic systems, these regulatory sequences includethe dihydrofolate reductase gene that is amplified in the presence ofmethotrexate, and the metallothionein genes that are amplified withheavy metals. In these cases, the nucleotide sequence encoding thepolypeptide would be operably linked with the regulatory sequence.

Expression Vectors

The present invention also relates to recombinant expression vectorscomprising a polynucleotide of the present invention, a promoter, andtranscriptional and translational stop signals. The various nucleicacids and control sequences described herein may be joined together toproduce a recombinant expression vector that may include one or more(several) convenient restriction sites to allow for insertion orsubstitution of the nucleotide sequence encoding the polypeptide at suchsites. Alternatively, a polynucleotide sequence of the present inventionmay be expressed by inserting the nucleotide sequence or a nucleic acidconstruct comprising the sequence into an appropriate vector forexpression. In creating the expression vector, the coding sequence islocated in the vector so that the coding sequence is operably linkedwith the appropriate control sequences for expression.

The recombinant expression vector may be any vector (e.g., a plasmid orvirus) that can be conveniently subjected to recombinant DNA proceduresand can bring about expression of the nucleotide sequence. The choice ofthe vector will typically depend on the compatibility of the vector withthe host cell into which the vector is to be introduced. The vectors maybe linear or closed circular plasmids.

The vector may be an autonomously replicating vector, i.e., a vectorthat exists as an extrachromosomal entity, the replication of which isindependent of chromosomal replication, e.g., a plasmid, anextrachromosomal element, a minichromosome, or an artificial chromosome.The vector may contain any means for assuring self-replication.Alternatively, the vector may be one that, when introduced into the hostcell, is integrated into the genome and replicated together with thechromosome(s) into which it has been integrated. Furthermore, a singlevector or plasmid or two or more vectors or plasmids that togethercontain the total DNA to be introduced into the genome of the host cell,or a transposon, may be used.

The vectors of the present invention preferably contain one or more(several) selectable markers that permit easy selection of transformed,transfected, transduced, or the like cells. A selectable marker is agene the product of which provides for biocide or viral resistance,resistance to heavy metals, prototrophy to auxotrophs, and the like.

Examples of bacterial selectable markers are the dal genes from Bacillussubtilis or Bacillus licheniformis, or markers that confer antibioticresistance such as ampicillin, kanamycin, chloramphenicol, ortetracycline resistance. Suitable markers for yeast host cells are ADE2,HIS3, LEU2, LYS2, MET3, TRP1, and URA3. Selectable markers for use in afilamentous fungal host cell include, but are not limited to, amdS(acetamidase), argB (ornithine carbamoyltransferase), bar(phosphinothricin acetyltransferase), hph (hygromycinphosphotransferase), niaD (nitrate reductase), pyrG(orotidine-5′-phosphate decarboxylase), sC (sulfate adenyltransferase),and trpC (anthranilate synthase), as well as equivalents thereof.Preferred for use in an Aspergillus cell are the amdS and pyrG genes ofAspergillus nidulans or Aspergillus oryzae and the bar gene ofStreptomyces hygroscopicus.

The vectors of the present invention preferably contain an element(s)that permits integration of the vector into the host cell's genome orautonomous replication of the vector in the cell independent of thegenome.

For integration into the host cell genome, the vector may rely on thepolynucleotide's sequence encoding the polypeptide or any other elementof the vector for integration into the genome by homologous ornonhomologous recombination. Alternatively, the vector may containadditional nucleotide sequences for directing integration by homologousrecombination into the genome of the host cell at a precise location(s)in the chromosome(s). To increase the likelihood of integration at aprecise location, the integrational elements should preferably contain asufficient number of nucleic acids, such as 100 to 10,000 base pairs,preferably 400 to 10,000 base pairs, and most preferably 800 to 10,000base pairs, which have a high degree of sequence identity to thecorresponding target sequence to enhance the probability of homologousrecombination. The integrational elements may be any sequence that ishomologous with the target sequence in the genome of the host cell.Furthermore, the integrational elements may be non-encoding or encodingnucleotide sequences. On the other hand, the vector may be integratedinto the genome of the host cell by non-homologous recombination.

For autonomous replication, the vector may further comprise an origin ofreplication enabling the vector to replicate autonomously in the hostcell in question. The origin of replication may be any plasmidreplicator mediating autonomous replication that functions in a cell.The term “origin of replication” or “plasmid replicator” is definedherein as a nucleotide sequence that enables a plasmid or vector toreplicate in vivo.

Examples of bacterial origins of replication are the origins ofreplication of plasmids pBR322, pUC19, pACYC177, and pACYC184 permittingreplication in E. coli, and pUB110, pE194, pTA1060, and pAMβ1 permittingreplication in Bacillus.

Examples of origins of replication for use in a yeast host cell are the2 micron origin of replication, ARS1, ARS4, the combination of ARS1 andCEN3, and the combination of ARS4 and CEN6.

Examples of origins of replication useful in a filamentous fungal cellare AMA1 and ANS1 (Gems et al., 1991, Gene 98: 61-67; Cullen et al.,1987, Nucleic Acids Research 15: 9163-9175; WO 00/24883). Isolation ofthe AMA1 gene and construction of plasmids or vectors comprising thegene can be accomplished according to the methods disclosed in WO00/24883.

More than one copy of a polynucleotide of the present invention may beinserted into a host cell to increase production of the gene product. Anincrease in the copy number of the polynucleotide can be obtained byintegrating at least one additional copy of the sequence into the hostcell genome or by including an amplifiable selectable marker gene withthe polynucleotide where cells containing amplified copies of theselectable marker gene, and thereby additional copies of thepolynucleotide, can be selected for by cultivating the cells in thepresence of the appropriate selectable agent.

The procedures used to ligate the elements described above to constructthe recombinant expression vectors of the present invention are wellknown to one skilled in the art (see, e.g., Sambrook et al., 1989,supra).

Host Cells

The present invention also relates to recombinant host cells, comprisingan isolated polynucleotide of the present invention operably linked toone or more (several) control sequences that direct the production of apolypeptide having feruloyl esterase activity. A construct or vectorcomprising a polynucleotide of the present invention is introduced intoa host cell so that the construct or vector is maintained as achromosomal integrant or as a self-replicating extra-chromosomal vectoras described earlier. The term “host cell” encompasses any progeny of aparent cell that is not identical to the parent cell due to mutationsthat occur during replication. The choice of a host cell will to a largeextent depend upon the gene encoding the polypeptide and its source.

The host cell may be any cell useful in the recombinant production of apolypeptide of the present invention, e.g., a prokaryote or a eukaryote.

The prokaryotic host cell may be any Gram positive bacterium or a Gramnegative bacterium. Gram positive bacteria include, but not limited to,Bacillus, Streptococcus, Streptomyces, Staphylococcus, Enterococcus,Lactobacillus, Lactococcus, Clostridium, Geobacillus, andOceanobacillus. Gram negative bacteria include, but not limited to, E.coli, Pseudomonas, Salmonella, Campylobacter, Helicobacter,Flavobacterium, Fusobacterium, Ilyobacter, Neisseria, and Ureaplasma.

The bacterial host cell may be any Bacillus cell. Bacillus cells usefulin the practice of the present invention include, but are not limitedto, Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis,Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacillusfirmus, Bacillus lautus, Bacillus lentus, Bacillus licheniformis,Bacillus megaterium, Bacillus pumilus, Bacillus stearothermophilus,Bacillus subtilis, and Bacillus thuringiensis cells.

In a preferred aspect, the bacterial host cell is a Bacillusamyloliquefaciens cell. In another preferred aspect, the bacterial hostcell is a Bacillus clausii cell. In another preferred aspect, thebacterial host cell is a Bacillus lentus cell. In another preferredaspect, the bacterial host cell is a Bacillus licheniformis cell. Inanother preferred aspect, the bacterial host cell is a Bacillusstearothermophilus cell. In another preferred aspect, the bacterial hostcell is a Bacillus subtilis cell.

The bacterial host cell may also be any Streptococcus cell.Streptococcus cells useful in the practice of the present inventioninclude, but are not limited to, Streptococcus equisimilis,Streptococcus pyogenes, Streptococcus uberis, and Streptococcus equisubsp. Zooepidemicus cells.

In a preferred aspect, the bacterial host cell is a Streptococcusequisimilis cell. In another preferred aspect, the bacterial host cellis a Streptococcus pyogenes cell. In another preferred aspect, thebacterial host cell is a Streptococcus uberis cell. In another preferredaspect, the bacterial host cell is a Streptococcus equi subsp.Zooepidemicus cell.

The bacterial host cell may also be any Streptomyces cell. Streptomycescells useful in the practice of the present invention include, but arenot limited to, Streptomyces achromogenes, Streptomyces avermitilis,Streptomyces coelicolor, Streptomyces griseus, and Streptomyces lividanscells.

In a preferred aspect, the bacterial host cell is a Streptomycesachromogenes cell. In another preferred aspect, the bacterial host cellis a Streptomyces avermitilis cell. In another preferred aspect, thebacterial host cell is a Streptomyces coelicolor cell. In anotherpreferred aspect, the bacterial host cell is a Streptomyces griseuscell. In another preferred aspect, the bacterial host cell is aStreptomyces lividans cell.

The introduction of DNA into a Bacillus cell may, for instance, beeffected by protoplast transformation (see, e.g., Chang and Cohen, 1979,Molecular General Genetics 168: 111-115), by using competent cells (see,e.g., Young and Spizizen, 1961, Journal of Bacteriology 81: 823-829, orDubnau and Davidoff-Abelson, 1971, Journal of Molecular Biology 56:209-221), by electroporation (see, e.g., Shigekawa and Dower, 1988,Biotechniques 6: 742-751), or by conjugation (see, e.g., Koehler andThorne, 1987, Journal of Bacteriology 169: 5271-5278). The introductionof DNA into an E coli cell may, for instance, be effected by protoplasttransformation (see, e.g., Hanahan, 1983, J. Mol. Biol. 166: 557-580) orelectroporation (see, e.g., Dower et al., 1988, Nucleic Acids Res. 16:6127-6145). The introduction of DNA into a Streptomyces cell may, forinstance, be effected by protoplast transformation and electroporation(see, e.g., Gong et al., 2004, Folia Microbiol. (Praha) 49: 399-405), byconjugation (see, e.g., Mazodier et al., 1989, J. Bacteriol. 171:3583-3585), or by transduction (see, e.g., Burke et al., 2001, Proc.Natl. Acad. Sci. USA 98: 6289-6294). The introduction of DNA into aPseudomonas cell may, for instance, be effected by electroporation (see,e.g., Choi et al., 2006, J. Microbiol. Methods 64: 391-397) or byconjugation (see, e.g., Pinedo and Smets, 2005, Appl. Environ.Microbiol. 71: 51-57). The introduction of DNA into a Streptococcus cellmay, for instance, be effected by natural competence (see, e.g., Perryand Kuramitsu, 1981, Infect. Immun. 32: 1295-1297), by protoplasttransformation (see, e.g., Catt and Jollick, 1991, Microbios. 68:189-207, by electroporation (see, e.g., Buckley et al., 1999, Appl.Environ. Microbiol. 65: 3800-3804) or by conjugation (see, e.g.,Clewell, 1981, Microbiol. Rev. 45: 409-436). However, any method knownin the art for introducing DNA into a host cell can be used.

The host cell may also be a eukaryote, such as a mammalian, insect,plant, or fungal cell.

In a preferred aspect, the host cell is a fungal cell. “Fungi” as usedherein includes the phyla Ascomycota, Basidiomycota, Chytridiomycota,and Zygomycota (as defined by Hawksworth et al., In, Ainsworth andBisby's Dictionary of The Fungi, 8th edition, 1995, CAB International,University Press, Cambridge, UK) as well as the Oomycota (as cited inHawksworth et al., 1995, supra, page 171) and all mitosporic fungi(Hawksworth et al., 1995, supra).

In a more preferred aspect, the fungal host cell is a yeast cell.“Yeast” as used herein includes ascosporogenous yeast (Endomycetales),basidiosporogenous yeast, and yeast belonging to the Fungi Imperfecti(Blastomycetes). Since the classification of yeast may change in thefuture, for the purposes of this invention, yeast shall be defined asdescribed in Biology and Activities of Yeast (Skinner, F. A., Passmore,S. M., and Davenport, R. R., eds, Soc. App. Bacteriol. Symposium SeriesNo. 9, 1980).

In an even more preferred aspect, the yeast host cell is a Candida,Hansenula, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, orYarrowia cell.

In a most preferred aspect, the yeast host cell is a Saccharomycescarlsbergensis cell. In another most preferred aspect, the yeast hostcell is a Saccharomyces cerevisiae cell. In another most preferredaspect, the yeast host cell is a Saccharomyces diastaticus cell. Inanother most preferred aspect, the yeast host cell is a Saccharomycesdouglasii cell. In another most preferred aspect, the yeast host cell isa Saccharomyces kluyveri cell. In another most preferred aspect, theyeast host cell is a Saccharomyces norbensis cell. In another mostpreferred aspect, the yeast host cell is a Saccharomyces oviformis cell.In another most preferred aspect, the yeast host cell is a Kluyveromyceslactis cell. In another most preferred aspect, the yeast host cell is aYarrowia lipolytica cell.

In another more preferred aspect, the fungal host cell is a filamentousfungal cell. “Filamentous fungi” include all filamentous forms of thesubdivision Eumycota and Oomycota (as defined by Hawksworth et al.,1995, supra). The filamentous fungi are generally characterized by amycelial wall composed of chitin, cellulose, glucan, chitosan, mannan,and other complex polysaccharides. Vegetative growth is by hyphalelongation and carbon catabolism is obligately aerobic. In contrast,vegetative growth by yeasts such as Saccharomyces cerevisiae is bybudding of a unicellular thallus and carbon catabolism may befermentative.

In an even more preferred aspect, the filamentous fungal host cell is anAcremonium, Aspergillus, Aureobasidium, Bjerkandera, Ceriporiopsis,Chrysosporium, Coprinus, Coriolus, Cryptococcus, Filibasidium, Fusarium,Humicola, Magnaporthe, Mucor, Myceliophthora, Neocallimastix,Neurospora, Paecilomyces, Penicillium, Phanerochaete, Phlebia,Piromyces, Pleurotus, Schizophyllum, Talaromyces, Thermoascus,Thielavia, Tolypocladium, Trametes, or Trichoderma cell.

In a most preferred aspect, the filamentous fungal host cell is anAspergillus awamori, Aspergillus fumigatus, Aspergillus foetidus,Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger orAspergillus oryzae cell. In another most preferred aspect, thefilamentous fungal host cell is a Fusarium bactridioides, Fusariumcerealis, Fusarium crookwellense, Fusarium culmorum, Fusariumgraminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi,Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusariumsambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusariumsulphureum, Fusarium torulosum, Fusarium trichothecioides, or Fusariumvenenatum cell. In another most preferred aspect, the filamentous fungalhost cell is a Bjerkandera adusta, Ceriporiopsis aneirina, Ceriporiopsisaneirina, Ceriporiopsis caregiea, Ceriporiopsis gilvescens,Ceriporiopsis pannocinta, Ceriporiopsis rivulosa, Ceriporiopsis subrufa,Ceriporiopsis subvermispora, Chrysosporium keratinophilum, Chrysosporiumlucknowense, Chrysosporium tropicum, Chrysosporium merdarium,Chrysosporium inops, Chrysosporium pannicola, Chrysosporiumqueenslandicum, Chrysosporium zonatum, Coprinus cinereus, Coriolushirsutus, Humicola insolens, Humicola lanuginosa, Mucor miehei,Myceliophthora thermophila, Neurospora crassa, Penicillium purpurogenum,Phanerochaete chrysosporium, Phlebia radiata, Pleurotus eryngii,Thielavia terrestris, Trametes villosa, Trametes versicolor, Trichodermaharzianum, Trichoderma koningii, Trichoderma longibrachiatum,Trichoderma reesei, or Trichoderma viride cell.

In another most preferred aspect, the filamentous fungal host cell is anAspergillus niger cell. In another most preferred aspect, thefilamentous fungal host cell is an Aspergillus oryzae cell. In anothermost preferred aspect, the filamentous fungal host cell is aChrysosporium lucknowense cell. In another most preferred aspect, thefilamentous fungal host cell is a Fusarium venenatum cell. In anothermost preferred aspect, the filamentous fungal host cell is aMyceliophthora thermophila cell. In another most preferred aspect, thefilamentous fungal host cell is a Trichoderma reesei cell.

Fungal cells may be transformed by a process involving protoplastformation, transformation of the protoplasts, and regeneration of thecell wall in a manner known per se. Suitable procedures fortransformation of Aspergillus and Trichoderma host cells are describedin EP 238 023 and Yelton et al., 1984, Proceedings of the NationalAcademy of Sciences USA 81: 1470-1474. Suitable methods for transformingFusarium species are described by Malardier et al., 1989, Gene 78:147-156, and WO 96/00787. Yeast may be transformed using the proceduresdescribed by Becker and Guarente, In Abelson, J. N. and Simon, M. I.,editors, Guide to Yeast Genetics and Molecular Biology, Methods inEnzymology, Volume 194, pp 182-187, Academic Press, Inc., New York; Itoet al., 1983, Journal of Bacteriology 153: 163; and Hinnen et al., 1978,Proceedings of the National Academy of Sciences USA 75: 1920.

Methods of Production

The present invention also relates to methods of producing a polypeptideof the present invention, comprising: (a) cultivating a cell, which inits wild-type form produces the polypeptide, under conditions conducivefor production of the polypeptide; and (b) recovering the polypeptide.In a preferred aspect, the cell is of the genus Thielavia. In a morepreferred aspect, the cell is Thielavia terrestris. In a most preferredaspect, the cell is Thielavia terrestris NRRL 8126.

The present invention also relates to methods of producing a polypeptideof the present invention, comprising: (a) cultivating a recombinant hostcell, as described herein, under conditions conducive for production ofthe polypeptide; and (b) recovering the polypeptide.

The present invention also relates to methods of producing a polypeptideof the present invention, comprising: (a) cultivating a recombinant hostcell under conditions conducive for production of the polypeptide,wherein the host cell comprises a mutant nucleotide sequence having atleast one mutation in the mature polypeptide coding sequence of SEQ IDNO: 1, wherein the mutant nucleotide sequence encodes a polypeptide thatcomprises or consists of the mature polypeptide of SEQ ID NO: 2; and (b)recovering the polypeptide.

In the production methods of the present invention, the cells arecultivated in a nutrient medium suitable for production of thepolypeptide using methods well known in the art. For example, the cellmay be cultivated by shake flask cultivation, and small-scale orlarge-scale fermentation (including continuous, batch, fed-batch, orsolid state fermentations) in laboratory or industrial fermentorsperformed in a suitable medium and under conditions allowing thepolypeptide to be expressed and/or isolated. The cultivation takes placein a suitable nutrient medium comprising carbon and nitrogen sources andinorganic salts, using procedures known in the art. Suitable media areavailable from commercial suppliers or may be prepared according topublished compositions (e.g., in catalogues of the American Type CultureCollection). If the polypeptide is secreted into the nutrient medium,the polypeptide can be recovered directly from the medium. If thepolypeptide is not secreted into the medium, it can be recovered fromcell lysates.

The polypeptides may be detected using methods known in the art that arespecific for the polypeptides. These detection methods may include useof specific antibodies, formation of an enzyme product, or disappearanceof an enzyme substrate. For example, an enzyme assay may be used todetermine the activity of the polypeptide as described herein.

The resulting polypeptide may be recovered using methods known in theart. For example, the polypeptide may be recovered from the nutrientmedium by conventional procedures including, but not limited to,centrifugation, filtration, extraction, spray-drying, evaporation, orprecipitation.

The polypeptides of the present invention may be purified by a varietyof procedures known in the art including, but not limited to,chromatography (e.g., ion exchange, affinity, hydrophobic,chromatofocusing, and size exclusion), electrophoretic procedures (e.g.,preparative isoelectric focusing), differential solubility (e.g.,ammonium sulfate precipitation), SDS-PAGE, or extraction (see, e.g.,Protein Purification, J.-C. Janson and Lars Ryden, editors, VCHPublishers, New York, 1989) to obtain substantially pure polypeptides.

Plants

The present invention also relates to plants, e.g., a transgenic plant,plant part, or plant cell, comprising an isolated polynucleotideencoding a polypeptide having feruloyl esterase activity of the presentinvention so as to express and produce the polypeptide in recoverablequantities. The polypeptide may be recovered from the plant or plantpart. Alternatively, the plant or plant part containing the recombinantpolypeptide may be used as such for improving the quality of a food orfeed, e.g., improving nutritional value, palatability, and rheologicalproperties, or to destroy an antinutritive factor.

The transgenic plant can be dicotyledonous (a dicot) or monocotyledonous(a monocot). Examples of monocot plants are grasses, such as meadowgrass (blue grass, Poa), forage grass such as Festuca, Lolium, temperategrass, such as Agrostis, and cereals, e.g., wheat, oats, rye, barley,rice, sorghum, and maize (corn).

Examples of dicot plants are tobacco, legumes, such as lupins, potato,sugar beet, pea, bean and soybean, and cruciferous plants (familyBrassicaceae), such as cauliflower, rape seed, and the closely relatedmodel organism Arabidopsis thaliana.

Examples of plant parts are stem, callus, leaves, root, fruits, seeds,and tubers as well as the individual tissues comprising these parts,e.g., epidermis, mesophyll, parenchyme, vascular tissues, meristems.Specific plant cell compartments, such as chloroplasts, apoplasts,mitochondria, vacuoles, peroxisomes and cytoplasm are also considered tobe a plant part. Furthermore, any plant cell, whatever the tissueorigin, is considered to be a plant part. Likewise, plant parts such asspecific tissues and cells isolated to facilitate the utilisation of theinvention are also considered plant parts, e.g., embryos, endosperms,aleurone and seeds coats.

Also included within the scope of the present invention are the progenyof such plants, plant parts, and plant cells.

The transgenic plant or plant cell expressing a polypeptide of thepresent invention may be constructed in accordance with methods known inthe art. In short, the plant or plant cell is constructed byincorporating one or more (several) expression constructs encoding apolypeptide of the present invention into the plant host genome orchloroplast genome and propagating the resulting modified plant or plantcell into a transgenic plant or plant cell.

The expression construct is conveniently a nucleic acid construct thatcomprises a polynucleotide encoding a polypeptide of the presentinvention operably linked with appropriate regulatory sequences requiredfor expression of the nucleotide sequence in the plant or plant part ofchoice. Furthermore, the expression construct may comprise a selectablemarker useful for identifying host cells into which the expressionconstruct has been integrated and DNA sequences necessary forintroduction of the construct into the plant in question (the latterdepends on the DNA introduction method to be used).

The choice of regulatory sequences, such as promoter and terminatorsequences and optionally signal or transit sequences, is determined, forexample, on the basis of when, where, and how the polypeptide is desiredto be expressed. For instance, the expression of the gene encoding apolypeptide of the present invention may be constitutive or inducible,or may be developmental, stage or tissue specific, and the gene productmay be targeted to a specific tissue or plant part such as seeds orleaves. Regulatory sequences are, for example, described by Tague etal., 1988, Plant Physiology 86: 506.

For constitutive expression, the 35S-CaMV, the maize ubiquitin 1, andthe rice actin 1 promoter may be used (Franck et al., 1980, Cell 21:285-294; Christensen et al., 1992, Plant Mol. Biol. 18: 675-689; Zhanget al., 1991, Plant Cell 3: 1155-1165). Organ-specific promoters may be,for example, a promoter from storage sink tissues such as seeds, potatotubers, and fruits (Edwards and Coruzzi, 1990, Ann. Rev. Genet. 24:275-303), or from metabolic sink tissues such as meristems (Ito et al.,1994, Plant Mol. Biol. 24: 863-878), a seed specific promoter such asthe glutelin, prolamin, globulin, or albumin promoter from rice (Wu etal., 1998, Plant and Cell Physiology 39: 885-889), a Vicia faba promoterfrom the legumin B4 and the unknown seed protein gene from Vicia faba(Conrad et al., 1998, Journal of Plant Physiology 152: 708-711), apromoter from a seed oil body protein (Chen et al., 1998, Plant and CellPhysiology 39: 935-941), the storage protein napA promoter from Brassicanapus, or any other seed specific promoter known in the art, e.g., asdescribed in WO 91/14772. Furthermore, the promoter may be a leafspecific promoter such as the rbcs promoter from rice or tomato (Kyozukaet al., 1993, Plant Physiology 102: 991-1000, the chlorella virusadenine methyltransferase gene promoter (Mitra and Higgins, 1994, PlantMolecular Biology 26: 85-93), or the aldP gene promoter from rice(Kagaya et al., 1995, Molecular and General Genetics 248: 668-674), or awound inducible promoter such as the potato pin2 promoter (Xu et al.,1993, Plant Molecular Biology 22: 573-588). Likewise, the promoter mayinducible by abiotic treatments such as temperature, drought, oralterations in salinity or induced by exogenously applied substancesthat activate the promoter, e.g., ethanol, oestrogens, plant hormonessuch as ethylene, abscisic acid, and gibberellic acid, and heavy metals.

A promoter enhancer element may also be used to achieve higherexpression of a polypeptide of the present invention in the plant. Forinstance, the promoter enhancer element may be an intron that is placedbetween the promoter and the nucleotide sequence encoding a polypeptideof the present invention. For instance, Xu et al., 1993, supra, disclosethe use of the first intron of the rice actin 1 gene to enhanceexpression.

The selectable marker gene and any other parts of the expressionconstruct may be chosen from those available in the art.

The nucleic acid construct is incorporated into the plant genomeaccording to conventional techniques known in the art, includingAgrobacterium-mediated transformation, virus-mediated transformation,microinjection, particle bombardment, biolistic transformation, andelectroporation (Gasser et al., 1990, Science 244: 1293; Potrykus, 1990,Bio/Technology 8: 535; Shimamoto et al., 1989, Nature 338: 274).

Presently, Agrobacterium tumefaciens-mediated gene transfer is themethod of choice for generating transgenic dicots (for a review, seeHooykas and Schilperoort, 1992, Plant Molecular Biology 19: 15-38) andcan also be used for transforming monocots, although othertransformation methods are often used for these plants. Presently, themethod of choice for generating transgenic monocots is particlebombardment (microscopic gold or tungsten particles coated with thetransforming DNA) of embryonic calli or developing embryos (Christou,1992, Plant Journal 2: 275-281; Shimamoto, 1994, Current OpinionBiotechnology 5: 158-162; Vasil et al., 1992, Bio/Technology 10:667-674). An alternative method for transformation of monocots is basedon protoplast transformation as described by Omirulleh et al., 1993,Plant Molecular Biology 21: 415-428. Additional transformation methodsfor use in accordance with the present disclosure include thosedescribed in U.S. Pat. Nos. 6,395,966 and 7,151,204 (both of which areherein incorporated by reference in their entirety).

Following transformation, the transformants having incorporated theexpression construct are selected and regenerated into whole plantsaccording to methods well-known in the art. Often the transformationprocedure is designed for the selective elimination of selection geneseither during regeneration or in the following generations by using, forexample, co-transformation with two separate T-DNA constructs or sitespecific excision of the selection gene by a specific recombinase.

The present invention also relates to methods of producing a polypeptideof the present invention comprising: (a) cultivating a transgenic plantor a plant cell comprising a polynucleotide encoding the polypeptidehaving feruloyl esterase activity of the present invention underconditions conducive for production of the polypeptide; and (b)recovering the polypeptide.

In embodiments, in addition to direct transformation of a particularplant genotype with a construct prepared according to the presentinvention, transgenic plants may be made by crossing a plant having aconstruct of the present invention to a second plant lacking theconstruct. For example, a construct encoding a polypeptide havingferuloyl esterase activity or a portion thereof can be introduced into aparticular plant variety by crossing, without the need for ever directlytransforming a plant of that given variety. Therefore, the presentinvention not only encompasses a plant directly regenerated from cellswhich have been transformed in accordance with the present invention,but also the progeny of such plants. As used herein, progeny may referto the offspring of any generation of a parent plant prepared inaccordance with the present invention. Such progeny may include a DNAconstruct prepared in accordance with the present invention, or aportion of a DNA construct prepared in accordance with the presentinvention. In embodiments, crossing results in a transgene of thepresent invention being introduced into a plant line by crosspollinating a starting line with a donor plant line that includes atransgene of the present invention. Non-limiting examples of such stepsare further articulated in U.S. Pat. No. 7,151,204.

It is envisioned that plants including a polypeptide having feruloylesterase activity of the present invention include plants generatedthrough a process of backcross conversion. For examples, plants of thepresent invention include plants referred to as a backcross convertedgenotype, line, inbred, or hybrid.

In embodiments, genetic markers may be used to assist in theintrogression of one or more transgenes of the invention from onegenetic background into another. Marker assisted selection offersadvantages relative to conventional breeding in that it can be used toavoid errors caused by phenotypic variations. Further, genetic markersmay provide data regarding the relative degree of elite germplasm in theindividual progeny of a particular cross. For example, when a plant witha desired trait which otherwise has a non-agronomically desirablegenetic background is crossed to an elite parent, genetic markers may beused to select progeny which not only possess the trait of interest, butalso have a relatively large proportion of the desired germplasm. Inthis way, the number of generations required to introgress one or moretraits into a particular genetic background is minimized.

Removal or Reduction of Feruloyl Esterase Activity

The present invention also relates to methods of producing a mutant of aparent cell, which comprises disrupting or deleting a polynucleotide, ora portion thereof, encoding a polypeptide of the present invention,which results in the mutant cell producing less of the polypeptide thanthe parent cell when cultivated under the same conditions.

The mutant cell may be constructed by reducing or eliminating expressionof a nucleotide sequence encoding a polypeptide of the present inventionusing methods well known in the art, for example, insertions,disruptions, replacements, or deletions. In a preferred aspect, thenucleotide sequence is inactivated. The nucleotide sequence to bemodified or inactivated may be, for example, the coding region or a partthereof essential for activity, or a regulatory element required for theexpression of the coding region. An example of such a regulatory orcontrol sequence may be a promoter sequence or a functional partthereof, i.e., a part that is sufficient for affecting expression of thenucleotide sequence. Other control sequences for possible modificationinclude, but are not limited to, a leader, polyadenylation sequence,propeptide sequence, signal peptide sequence, transcription terminator,and transcriptional activator.

Modification or inactivation of the nucleotide sequence may be performedby subjecting the parent cell to mutagenesis and selecting for mutantcells in which expression of the nucleotide sequence has been reduced oreliminated. The mutagenesis, which may be specific or random, may beperformed, for example, by use of a suitable physical or chemicalmutagenizing agent, by use of a suitable oligonucleotide, or bysubjecting the DNA sequence to PCR generated mutagenesis. Furthermore,the mutagenesis may be performed by use of any combination of thesemutagenizing agents.

Examples of a physical or chemical mutagenizing agent suitable for thepresent purpose include ultraviolet (UV) irradiation, hydroxylamine,N-methyl-N′-nitro-N-nitrosoguanidine (MNNG), O-methyl hydroxylamine,nitrous acid, ethyl methane sulphonate (EMS), sodium bisulphite, formicacid, and nucleotide analogues.

When such agents are used, the mutagenesis is typically performed byincubating the parent cell to be mutagenized in the presence of themutagenizing agent of choice under suitable conditions, and screeningand/or selecting for mutant cells exhibiting reduced or no expression ofthe gene.

Modification or inactivation of the nucleotide sequence may beaccomplished by introduction, substitution, or removal of one or more(several) nucleotides in the gene or a regulatory element required forthe transcription or translation thereof. For example, nucleotides maybe inserted or removed so as to result in the introduction of a stopcodon, the removal of the start codon, or a change in the open readingframe. Such modification or inactivation may be accomplished bysite-directed mutagenesis or PCR generated mutagenesis in accordancewith methods known in the art. Although, in principle, the modificationmay be performed in vivo, i.e., directly on the cell expressing thenucleotide sequence to be modified, it is preferred that themodification be performed in vitro as exemplified below.

An example of a convenient way to eliminate or reduce expression of anucleotide sequence by a cell is based on techniques of genereplacement, gene deletion, or gene disruption. For example, in the genedisruption method, a nucleic acid sequence corresponding to theendogenous nucleotide sequence is mutagenized in vitro to produce adefective nucleic acid sequence that is then transformed into the parentcell to produce a defective gene. By homologous recombination, thedefective nucleic acid sequence replaces the endogenous nucleotidesequence. It may be desirable that the defective nucleotide sequencealso encodes a marker that may be used for selection of transformants inwhich the nucleotide sequence has been modified or destroyed. In aparticularly preferred aspect, the nucleotide sequence is disrupted witha selectable marker such as those described herein.

Alternatively, modification or inactivation of the nucleotide sequencemay be performed by established anti-sense or RNAi techniques using asequence complementary to the nucleotide sequence. More specifically,expression of the nucleotide sequence by a cell may be reduced oreliminated by introducing a sequence complementary to the nucleotidesequence of the gene that may be transcribed in the cell and is capableof hybridizing to the mRNA produced in the cell. Under conditionsallowing the complementary anti-sense nucleotide sequence to hybridizeto the mRNA, the amount of protein translated is thus reduced oreliminated.

The present invention further relates to a mutant cell of a parent cellthat comprises a disruption or deletion of a nucleotide sequenceencoding the polypeptide or a control sequence thereof, which results inthe mutant cell producing less of the polypeptide or no polypeptidecompared to the parent cell.

The polypeptide-deficient mutant cells so created are particularlyuseful as host cells for the expression of native and/or heterologouspolypeptides. Therefore, the present invention further relates tomethods of producing a native or heterologous polypeptide, comprising:(a) cultivating the mutant cell under conditions conducive forproduction of the polypeptide; and (b) recovering the polypeptide. Theterm “heterologous polypeptides” is defined herein as polypeptides thatare not native to the host cell, a native protein in which modificationshave been made to alter the native sequence, or a native protein whoseexpression is quantitatively altered as a result of a manipulation ofthe host cell by recombinant DNA techniques.

In a further aspect, the present invention relates to a method ofproducing a protein product essentially free of feruloyl esteraseactivity by fermentation of a cell that produces both a polypeptide ofthe present invention as well as the protein product of interest byadding an effective amount of an agent capable of inhibiting feruloylesterase activity to the fermentation broth before, during, or after thefermentation has been completed, recovering the product of interest fromthe fermentation broth, and optionally subjecting the recovered productto further purification.

In a further aspect, the present invention relates to a method ofproducing a protein product essentially free of feruloyl esteraseactivity by cultivating the cell under conditions permitting theexpression of the product, subjecting the resultant culture broth to acombined pH and temperature treatment so as to reduce the feruloylesterase activity substantially, and recovering the product from theculture broth. Alternatively, the combined pH and temperature treatmentmay be performed on an enzyme preparation recovered from the culturebroth. The combined pH and temperature treatment may optionally be usedin combination with a treatment with an feruloyl esterase inhibitor.

In accordance with this aspect of the invention, it is possible toremove at least 60%, preferably at least 75%, more preferably at least85%, still more preferably at least 95%, and most preferably at least99% of the feruloyl esterase activity. Complete removal of feruloylesterase activity may be obtained by use of this method.

The combined pH and temperature treatment is preferably carried out at apH in the range of 2-4 or 9-11 and a temperature in the range of atleast 60-70° C. for a sufficient period of time to attain the desiredeffect, where typically, 30 to 60 minutes is sufficient.

The methods used for cultivation and purification of the product ofinterest may be performed by methods known in the art.

The methods of the present invention for producing an essentiallyferuloyl esterase-free product is of particular interest in theproduction of eukaryotic polypeptides, in particular fungal proteinssuch as enzymes. The enzyme may be selected from, e.g., an amylolyticenzyme, lipolytic enzyme, proteolytic enzyme, cellulolytic enzyme,oxidoreductase, or plant cell-wall degrading enzyme. Examples of suchenzymes include an aminopeptidase, amylase, amyloglucosidase,carbohydrase, carboxypeptidase, catalase, cellobiohydrolase, cellulase,chitinase, cutinase, cyclodextrin glycosyltransferase,deoxyribonuclease, endoglucanase, esterase, galactosidase,beta-galactosidase, glucoamylase, glucose oxidase, glucosidase,haloperoxidase, hemicellulase, invertase, isomerase, laccase, ligase,lipase, lyase, mannosidase, oxidase, pectinolytic enzyme, peroxidase,phytase, phenoloxidase, polyphenoloxidase, proteolytic enzyme,ribonuclease, transferase, transglutaminase, or xylanase. The feruloylesterase-deficient cells may also be used to express heterologousproteins of pharmaceutical interest such as hormones, growth factors,receptors, and the like.

It will be understood that the term “eukaryotic polypeptides” includesnot only native polypeptides, but also those polypeptides, e.g.,enzymes, which have been modified by amino acid substitutions, deletionsor additions, or other such modifications to enhance activity,thermostability, pH tolerance and the like.

In a further aspect, the present invention relates to a protein productessentially free from feruloyl esterase activity that is produced by amethod of the present invention.

Methods of Inhibiting Expression of a Polypeptide Having FeruloylEsterase Activity

The present invention also relates to methods of inhibiting theexpression of a polypeptide having feruloyl esterase activity in a cell,comprising administering to the cell or expressing in the cell adouble-stranded RNA (dsRNA) molecule, wherein the dsRNA comprises asubsequence of a polynucleotide of the present invention. In a preferredaspect, the dsRNA is about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 ormore duplex nucleotides in length.

The dsRNA is preferably a small interfering RNA (sRNA) or a micro RNA(miRNA). In a preferred aspect, the dsRNA is small interfering RNA(siRNAs) for inhibiting transcription. In another preferred aspect, thedsRNA is micro RNA (miRNAs) for inhibiting translation.

The present invention also relates to such double-stranded RNA (dsRNA)molecules, comprising a portion of the mature polypeptide codingsequence of SEQ ID NO: 1 for inhibiting expression of a polypeptide in acell. While the present invention is not limited by any particularmechanism of action, the dsRNA can enter a cell and cause thedegradation of a single-stranded RNA (ssRNA) of similar or identicalsequences, including endogenous mRNAs. When a cell is exposed to dsRNA,mRNA from the homologous gene is selectively degraded by a processcalled RNA interference (RNAi).

The dsRNAs of the present invention can be used in gene-silencing. Inone aspect, the invention provides methods to selectively degrade RNAusing the dsRNAis of the present invention. The process may be practicedin vitro, ex vivo or in vivo. In one aspect, the dsRNA molecules can beused to generate a loss-of-function mutation in a cell, an organ or ananimal. Methods for making and using dsRNA molecules to selectivelydegrade RNA are well known in the art, see, for example, U.S. Pat. No.6,506,559; U.S. Pat. No. 6,511,824; U.S. Pat. No. 6,515,109; and U.S.Pat. No. 6,489,127.

Compositions

The present invention also relates to compositions comprising apolypeptide of the present invention. Preferably, the compositions areenriched in such a polypeptide. The term “enriched” indicates that theferuloyl esterase activity of the composition has been increased, e.g.,with an enrichment factor of at least 1.1.

The composition may comprise a polypeptide of the present invention asthe major enzymatic component, e.g., a mono-component composition.Alternatively, the composition may comprise multiple enzymaticactivities, such as an aminopeptidase, amylase, carbohydrase,carboxypeptidase, catalase, cellulase, chitinase, cutinase, cyclodextringlycosyltransferase, deoxyribonuclease, esterase, alpha-galactosidase,beta-galactosidase, glucoamylase, alpha-glucosidase, beta-glucosidase,haloperoxidase, invertase, laccase, lipase, mannosidase, oxidase,pectinolytic enzyme, peptidoglutaminase, peroxidase, phytase,polyphenoloxidase, proteolytic enzyme, ribonuclease, transglutaminase,or xylanase. The additional enzyme(s) may be produced, for example, by amicroorganism belonging to the genus Aspergillus, preferably Aspergillusaculeatus, Aspergillus awamori, Aspergillus fumigatus, Aspergillusfoetidus, Aspergillus japonicus, Aspergillus nidulans, Aspergillusniger, or Aspergillus oryzae; Fusarium, preferably Fusariumbactridioides, Fusarium cerealis, Fusarium crookwellense, Fusariumculmorum, Fusarium graminearum, Fusarium graminum, Fusariumheterosporum, Fusarium negundi, Fusarium oxysporum, Fusariumreticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum,Fusarium sulphureum, Fusarium toruloseum, Fusarium trichothecioides, orFusarium venenatum; Humicola, preferably Humicola insolens or Humicolalanuginosa; or Trichoderma, preferably Trichoderma harzianum,Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei,or Trichoderma viride.

The polypeptide compositions may be prepared in accordance with methodsknown in the art and may be in the form of a liquid or a drycomposition. For instance, the polypeptide composition may be in theform of a granulate or a microgranulate. The polypeptide to be includedin the composition may be stabilized in accordance with methods known inthe art.

Examples are given below of preferred uses of the polypeptidecompositions of the invention. The dosage of the polypeptide compositionof the invention and other conditions under which the composition isused may be determined on the basis of methods known in the art.

Uses

The present invention is also directed to methods of using thepolypeptides having feruloyl esterase activity, or compositions thereof.The polypeptides of the present invention can be used for degrading orconverting plant cell walls or any xylan-containing material, e.g.,lignocellulose, originating from plant cells walls (see, for example, WO2002/18561). Examples of various uses are described below. The dosage ofthe polypeptides of the present invention and other conditions underwhich the polypeptides are used may be determined on the basis ofmethods known in the art.

The enzymatic degradation of a xylan-containing material is facilitatedby full or partial removal of the side branches. The polypeptides of thepresent invention are preferably used in conjunction with other xylandegrading enzymes such as xylanases, acetylxylan esterases,arabinofuranosidases, xylosidases, feruloyl esterases, glucuronidases,and a combination thereof, in processes wherein xylan-containingmaterial is to be degraded. For example, acetyl groups can be removed byacetylxylan esterases; arabinose groups by alpha-arabinosidases;feruloyl groups by feruloyl esterases, and glucuronic acid groups byalpha-glucuronidases. The oligomers released by the xylanases, or by acombination of xylanases and side branch-hydrolyzing enzymes, can befurther degraded to free xylose by beta-xylosidases.

The present invention also relates to methods for degrading orconverting a cellulosic or xylan-containing material, comprising:treating the cellulosic or xylan-containing material with an enzymecomposition in the presence of a polypeptide having feruloyl esteraseactivity of the present invention. In a preferred aspect, the methodfurther comprises recovering the degraded or converted cellulosic orxylan-containing material.

The present invention also relates to methods for producing afermentation product, comprising: (a) saccharifying a cellulosic orxylan-containing material with an enzyme composition in the presence ofa polypeptide having feruloyl esterase activity of the presentinvention; (b) fermenting the saccharified cellulosic orxylan-containing material with one or more (several) fermentingmicroorganisms to produce the fermentation product; and (c) recoveringthe fermentation product from the fermentation.

The present invention also relates to methods of fermenting a cellulosicor xylan-containing material, comprising: fermenting the cellulosic orxylan-containing material with one or more (several) fermentingmicroorganisms, wherein the cellulosic or xylan-containing material issaccharified with an enzyme composition in the presence of a polypeptidehaving feruloyl esterase activity of the present invention. In apreferred aspect, the fermenting of the cellulosic or xylan-containingmaterial produces a fermentation product. In another preferred aspect,the method further comprises recovering the fermentation product fromthe fermentation.

The methods of the present invention can be used to saccharify acellulosic or xylan-containing material to fermentable sugars andconvert the fermentable sugars to many useful substances, e.g., fuel,potable ethanol, and/or fermentation products (e.g., acids, alcohols,ketones, gases, and the like). The production of a desired fermentationproduct from cellulosic or xylan-containing material typically involvespretreatment, enzymatic hydrolysis (saccharification), and fermentation.

The processing of cellulosic or xylan-containing material according tothe present invention can be accomplished using processes conventionalin the art. Moreover, the methods of the present invention can beimplemented using any conventional biomass processing apparatusconfigured to operate in accordance with the invention.

Hydrolysis (saccharification) and fermentation, separate orsimultaneous, include, but are not limited to, separate hydrolysis andfermentation (SHF); simultaneous saccharification and fermentation(SSF); simultaneous saccharification and cofermentation (SSCF); hybridhydrolysis and fermentation (HHF); separate hydrolysis andco-fermentation (SHCF); hybrid hydrolysis and fermentation (HHCF); anddirect microbial conversion (DMC). SHF uses separate process steps tofirst enzymatically hydrolyze cellulosic or xylan-containing material tofermentable sugars, e.g., glucose, cellobiose, cellotriose, and pentosesugars, and then ferment the fermentable sugars to ethanol. In SSF, theenzymatic hydrolysis of cellulosic or xylan-containing material and thefermentation of sugars to ethanol are combined in one step (Philippidis,G. P., 1996, Cellulose bioconversion technology, in Handbook onBioethanol: Production and Utilization, Wyman, C. E., ed., Taylor &Francis, Washington, D.C., 179-212). SSCF involves the cofermentation ofmultiple sugars (Sheehan, J., and Himmel, M., 1999, Enzymes, energy andthe environment: A strategic perspective on the U.S. Department ofEnergy's research and development activities for bioethanol, Biotechnol.Prog. 15: 817-827). HHF involves a separate hydrolysis step, and inaddition a simultaneous saccharification and hydrolysis step, which canbe carried out in the same reactor. The steps in an HHF process can becarried out at different temperatures, i.e., high temperature enzymaticsaccharification followed by SSF at a lower temperature that thefermentation strain can tolerate. DMC combines all three processes(enzyme production, hydrolysis, and fermentation) in one or more stepswhere the same organism is used to produce the enzymes for conversion ofthe cellulosic or xylan-containing material to fermentable sugars and toconvert the fermentable sugars into a final product (Lynd, L. R.,Weimer, P. J., van Zyl, W. H., and Pretorius, I. S., 2002, Microbialcellulose utilization: Fundamentals and biotechnology, Microbiol. Mol.Biol. Reviews 66: 506-577). It is understood herein that any methodknown in the art comprising pretreatment, enzymatic hydrolysis(saccharification), fermentation, or a combination thereof can be usedin the practicing the methods of the present invention.

A conventional apparatus can include a fed-batch stirred reactor, abatch stirred reactor, a continuous flow stirred reactor withultrafiltration, and/or a continuous plug-flow column reactor (Fernandade Castilhos Corazza, Flávio Faria de Moraes, Gisella Maria Zanin andIvo Neitzel, 2003, Optimal control in fed-batch reactor for thecellobiose hydrolysis, Acta Scientiarum. Technology 25: 33-38; Gusakov,A. V., and Sinitsyn, A. P., 1985, Kinetics of the enzymatic hydrolysisof cellulose: 1. A mathematical model for a batch reactor process, Enz.Microb. Technol. 7: 346-352), an attrition reactor (Ryu, S. K., and Lee,J. M., 1983, Bioconversion of waste cellulose by using an attritionbioreactor, Biotechnol. Bioeng. 25: 53-65), or a reactor with intensivestirring induced by an electromagnetic field (Gusakov, A. V., Sinitsyn,A. P., Davydkin, I. Y., Davydkin, V. Y., Protas, O. V., 1996,Enhancement of enzymatic cellulose hydrolysis using a novel type ofbioreactor with intensive stirring induced by electromagnetic field,Appl. Biochem. Biotechnol. 56: 141-153). Additional reactor typesinclude: fluidized bed, upflow blanket, immobilized, and extruder typereactors for hydrolysis and/or fermentation.

Pretreatment. In practicing the methods of the present invention, anypretreatment process known in the art can be used to disrupt plant cellwall components of cellulosic and/or xylan-containing material (Chandraet al., 2007, Substrate pretreatment: The key to effective enzymatichydrolysis of lignocellulosics? Adv. Biochem. Engin./Biotechnol. 108:67-93; Galbe and Zacchi, 2007, Pretreatment of lignocellulosic materialsfor efficient bioethanol production, Adv. Biochem. Engin./Biotechnol.108: 41-65; Hendriks and Zeeman, 2009, Pretreatments to enhance thedigestibility of lignocellulosic biomass, Bioresource Technol. 100:10-18; Mosier et al., 2005, Features of promising technologies forpretreatment of lignocellulosic biomass, Bioresource Technol. 96:673-686; Taherzadeh and Karimi, 2008, Pretreatment of lignocellulosicwastes to improve ethanol and biogas production: A review, Int. J. ofMol. Sci. 9: 1621-1651; Yang and Wyman, 2008, Pretreatment: the key tounlocking low-cost cellulosic ethanol, Biofuels Bioproducts andBiorefining-Biofpr. 2: 26-40).

Cellulosic or xylan-containing material can also be subjected toparticle size reduction, pre-soaking, wetting, washing, or conditioningprior to pretreatment using methods known in the art.

Conventional pretreatments include, but are not limited to, steampretreatment (with or without explosion), dilute acid pretreatment, hotwater pretreatment, alkaline pretreatment, lime pretreatment, wetoxidation, wet explosion, ammonia fiber explosion, organosolvpretreatment, and biological pretreatment. Additional pretreatmentsinclude ammonia percolation, ultrasound, electroporation, microwave,supercritical CO₂, supercritical H₂O, ozone, and gamma irradiationpretreatments.

Cellulosic or xylan-containing material can be pretreated beforehydrolysis and/or fermentation. Pretreatment is preferably performedprior to the hydrolysis. Alternatively, the pretreatment can be carriedout simultaneously with enzyme hydrolysis to release fermentable sugars,such as glucose, xylose, and/or cellobiose. In most cases thepretreatment step itself results in some conversion of biomass tofermentable sugars (even in absence of enzymes).

Steam Pretreatment. In steam pretreatment, cellulosic orxylan-containing material is heated to disrupt the plant cell wallcomponents, including lignin, hemicellulose, and cellulose to make thecellulose and other fractions, e.g., hemicellulose, accessible toenzymes. Cellulosic or xylan-containing material is passed to or througha reaction vessel where steam is injected to increase the temperature tothe required temperature and pressure and is retained therein for thedesired reaction time. Steam pretreatment is preferably done at 140-230°C., more preferably 160-200° C., and most preferably 170-190° C., wherethe optimal temperature range depends on any addition of a chemicalcatalyst. Residence time for the steam pretreatment is preferably 1-15minutes, more preferably 3-12 minutes, and most preferably 4-10 minutes,where the optimal residence time depends on temperature range and anyaddition of a chemical catalyst. Steam pretreatment allows forrelatively high solids loadings, so that cellulosic or xylan-containingmaterial is generally only moist during the pretreatment. The steampretreatment is often combined with an explosive discharge of thematerial after the pretreatment, which is known as steam explosion, thatis, rapid flashing to atmospheric pressure and turbulent flow of thematerial to increase the accessible surface area by fragmentation (Duffand Murray, 1996, Bioresource Technology 855: 1-33; Galbe and Zacchi,2002, Appl. Microbiol. Biotechnol. 59: 618-628; U.S. Patent ApplicationNo. 20020164730). During steam pretreatment, hemicellulose acetyl groupsare cleaved and the resulting acid autocatalyzes partial hydrolysis ofthe hemicellulose to monosaccharides and oligosaccharides. Lignin isremoved to only a limited extent.

A catalyst such as H₂SO₄ or SO₂ (typically 0.3 to 3% w/w) is often addedprior to steam pretreatment, which decreases the time and temperature,increases the recovery, and improves enzymatic hydrolysis (Ballesteroset al., 2006, Appl. Biochem. Biotechnol. 129-132: 496-508; Varga et al.,2004, Appl. Biochem. Biotechnol. 113-116: 509-523; Sassner et al., 2006,Enzyme Microb. Technol. 39: 756-762).

Chemical Pretreatment: The term “chemical treatment” refers to anychemical pretreatment that promotes the separation and/or release ofcellulose, hemicellulose, and/or lignin. Examples of suitable chemicalpretreatment processes include, for example, dilute acid pretreatment,lime pretreatment, wet oxidation, ammonia fiber/freeze explosion (AFEX),ammonia percolation (APR), and organosolv pretreatments.

In dilute acid pretreatment, cellulosic or xylan-containing material ismixed with dilute acid, typically H₂SO₄, and water to form a slurry,heated by steam to the desired temperature, and after a residence timeflashed to atmospheric pressure. The dilute acid pretreatment can beperformed with a number of reactor designs, e.g., plug-flow reactors,counter-current reactors, or continuous counter-current shrinking bedreactors (Duff and Murray, 1996, supra; Schell et al., 2004, BioresourceTechnol. 91: 179-188; Lee et al., 1999, Adv. Biochem. Eng. Biotechnol.65: 93-115).

Several methods of pretreatment under alkaline conditions can also beused. These alkaline pretreatments include, but are not limited to, limepretreatment, wet oxidation, ammonia percolation (APR), and ammoniafiber/freeze explosion (AFEX).

Lime pretreatment is performed with calcium carbonate, sodium hydroxide,or ammonia at low temperatures of 85-150° C. and residence times from 1hour to several days (Wyman et al., 2005, Bioresource Technol. 96:1959-1966; Mosier et al., 2005, Bioresource Technol. 96: 673-686). WO2006/110891, WO 2006/11899, WO 2006/11900, and WO 2006/110901 disclosepretreatment methods using ammonia.

Wet oxidation is a thermal pretreatment performed typically at 180-200°C. for 5-15 minutes with addition of an oxidative agent such as hydrogenperoxide or over-pressure of oxygen (Schmidt and Thomsen, 1998,Bioresource Technol. 64: 139-151; Palonen et al., 2004, Appl. Biochem.Biotechnol. 117: 1-17; Varga et al., 2004, Biotechnol. Bioeng. 88:567-574; Martin et al., 2006, J. Chem. Technol. Biotechnol. 81:1669-1677). The pretreatment is performed at preferably 1-40% drymatter, more preferably 2-30% dry matter, and most preferably 5-20% drymatter, and often the initial pH is increased by the addition of alkalisuch as sodium carbonate.

A modification of the wet oxidation pretreatment method, known as wetexplosion (combination of wet oxidation and steam explosion), can handledry matter up to 30%. In wet explosion, the oxidizing agent isintroduced during pretreatment after a certain residence time. Thepretreatment is then ended by flashing to atmospheric pressure (WO2006/032282).

Ammonia fiber explosion (AFEX) involves treating cellulosic orxylan-containing material with liquid or gaseous ammonia at moderatetemperatures such as 90-100° C. and high pressure such as 17-20 bar for5-10 minutes, where the dry matter content can be as high as 60%(Gollapalli et al., 2002, Appl. Biochem. Biotechnol. 98: 23-35;Chundawat et al., 2007, Biotechnol. Bioeng. 96: 219-231; Alizadeh etal., 2005, Appl. Biochem. Biotechnol. 121: 1133-1141; Teymouri et al.,2005, Bioresource Technol. 96: 2014-2018). AFEX pretreatment results inthe depolymerization of cellulose and partial hydrolysis ofhemicellulose. Lignin-carbohydrate complexes are cleaved.

Organosolv pretreatment delignifies cellulosic or xylan-containingmaterial by extraction using aqueous ethanol (40-60% ethanol) at160-200° C. for 30-60 minutes (Pan et al., 2005, Biotechnol. Bioeng. 90:473-481; Pan et al., 2006, Biotechnol. Bioeng. 94: 851-861; Kurabi etal., 2005, Appl. Biochem. Biotechnol. 121: 219-230). Sulphuric acid isusually added as a catalyst. In organosolv pretreatment, the majority ofhemicellulose is removed.

Other examples of suitable pretreatment methods are described by Schellet al., 2003, Appl. Biochem. and Biotechnol. Vol. 105-108, p. 69-85, andMosier et al., 2005, Bioresource Technology 96: 673-686, and U.S.Published Application 2002/0164730.

In one aspect, the chemical pretreatment is preferably carried out as anacid treatment, and more preferably as a continuous dilute and/or mildacid treatment. The acid is typically sulfuric acid, but other acids canalso be used, such as acetic acid, citric acid, nitric acid, phosphoricacid, tartaric acid, succinic acid, hydrogen chloride, or mixturesthereof. Mild acid treatment is conducted in the pH range of preferably1-5, more preferably 1-4, and most preferably 1-3. In one aspect, theacid concentration is in the range from preferably 0.01 to 20 wt % acid,more preferably 0.05 to 10 wt % acid, even more preferably 0.1 to 5 wt %acid, and most preferably 0.2 to 2.0 wt % acid. The acid is contactedwith cellulosic or xylan-containing material and held at a temperaturein the range of preferably 160-220° C., and more preferably 165-195° C.,for periods ranging from seconds to minutes to, e.g., 1 second to 60minutes.

In another aspect, pretreatment is carried out as an ammonia fiberexplosion step (AFEX pretreatment step).

In another aspect, pretreatment takes place in an aqueous slurry. Inpreferred aspects, cellulosic or xylan-containing material is presentduring pretreatment in amounts preferably between 10-80 wt %, morepreferably between 20-70 wt %, and most preferably between 30-60 wt %,such as around 50 wt %. The pretreated cellulosic or xylan-containingmaterial can be unwashed or washed using any method known in the art,e.g., washed with water.

Mechanical Pretreatment: The term “mechanical pretreatment” refers tovarious types of grinding or milling (e.g., dry milling, wet milling, orvibratory ball milling).

Physical Pretreatment: The term “physical pretreatment” refers to anypretreatment that promotes the separation and/or release of cellulose,hemicellulose, and/or lignin from cellulosic or xylan-containingmaterial. For example, physical pretreatment can involve irradiation(e.g., microwave irradiation), steaming/steam explosion,hydrothermolysis, and combinations thereof.

Physical pretreatment can involve high pressure and/or high temperature(steam explosion). In one aspect, high pressure means pressure in therange of preferably about 300 to about 600 psi, more preferably about350 to about 550 psi, and most preferably about 400 to about 500 psi,such as around 450 psi. In another aspect, high temperature meanstemperatures in the range of about 100 to about 300° C., preferablyabout 140 to about 235° C. In a preferred aspect, mechanicalpretreatment is performed in a batch-process, steam gun hydrolyzersystem that uses high pressure and high temperature as defined above,e.g., a Sunds Hydrolyzer available from Sunds Defibrator AB, Sweden.

Combined Physical and Chemical Pretreatment: Cellulosic orxylan-containing material can be pretreated both physically andchemically. For instance, the pretreatment step can involve dilute ormild acid treatment and high temperature and/or pressure treatment. Thephysical and chemical pretreatments can be carried out sequentially orsimultaneously, as desired. A mechanical pretreatment can also beincluded.

Accordingly, in a preferred aspect, cellulosic or xylan-containingmaterial is subjected to mechanical, chemical, or physical pretreatment,or any combination thereof, to promote the separation and/or release ofcellulose, hemicellulose, and/or lignin.

Biological Pretreatment: The term “biological pretreatment” refers toany biological pretreatment that promotes the separation and/or releaseof cellulose, hemicellulose, and/or lignin from cellulosic orxylan-containing material. Biological pretreatment techniques caninvolve applying lignin-solubilizing microorganisms (see, for example,Hsu, T.-A., 1996, Pretreatment of biomass, in Handbook on Bioethanol:Production and Utilization, Wyman, C. E., ed., Taylor & Francis,Washington, D.C., 179-212; Ghosh and Singh, 1993, Physicochemical andbiological treatments for enzymatic/microbial conversion of cellulosicbiomass, Adv. Appl. Microbiol. 39: 295-333; McMillan, J. D., 1994,Pretreating lignocellulosic biomass: a review, in Enzymatic Conversionof Biomass for Fuels Production, Himmel, M. E., Baker, J. O., andOverend, R. P., eds., ACS Symposium Series 566, American ChemicalSociety, Washington, D.C., chapter 15; Gong, C. S., Cao, N. J., Du, J.,and Tsao, G. T., 1999, Ethanol production from renewable resources, inAdvances in Biochemical Engineering/Biotechnology, Scheper, T., ed.,Springer-Verlag Berlin Heidelberg, Germany, 65: 207-241; Olsson andHahn-Hagerdal, 1996, Fermentation of lignocellulosic hydrolysates forethanol production, Enz. Microb. Tech. 18: 312-331; and Vallander andEriksson, 1990, Production of ethanol from lignocellulosic materials:State of the art, Adv. Biochem. Eng./Biotechnol. 42: 63-95).

Saccharification. In the hydrolysis step, also known assaccharification, the pretreated cellulosic or xylan-containing materialis hydrolyzed to break down cellulose and hemicellulose to fermentablesugars, such as glucose, cellobiose, xylose, xylulose, arabinose,mannose, galactose, and/or soluble oligosaccharides. The hydrolysis isperformed enzymatically by an enzyme composition in the presence of apolypeptide having feruloyl esterase activity of the present invention.The components of the enzyme composition can also be added sequentially.

Enzymatic hydrolysis is preferably carried out in a suitable aqueousenvironment under conditions that can be readily determined by oneskilled in the art. In a preferred aspect, hydrolysis is performed underconditions suitable for the activity of the enzyme(s), i.e., optimal forthe enzyme(s). The hydrolysis can be carried out as a fed batch orcontinuous process where the pretreated cellulosic or xylan-containingmaterial (substrate) is fed gradually to, for example, an enzymecontaining hydrolysis solution.

The saccharification is generally performed in stirred-tank reactors orfermentors under controlled pH, temperature, and mixing conditions.Suitable process time, temperature and pH conditions can readily bedetermined by one skilled in the art. For example, the saccharificationcan last up to 200 hours, but is typically performed for preferablyabout 12 to about 96 hours, more preferably about 16 to about 72 hours,and most preferably about 24 to about 48 hours. The temperature is inthe range of preferably about 25° C. to about 70° C., more preferablyabout 30° C. to about 65° C., and more preferably about 40° C. to 60°C., in particular about 50° C. The pH is in the range of preferablyabout 3 to about 8, more preferably about 3.5 to about 7, and mostpreferably about 4 to about 6, in particular about pH 5. The dry solidscontent is in the range of preferably about 5 to about 50 wt %, morepreferably about 10 to about 40 wt %, and most preferably about 20 toabout 30 wt %.

The enzyme composition preferably comprises enzymes having cellulolyticactivity and/or xylan degrading activity. In one aspect, the enzymecomposition comprises one or more (several) xylan degrading enzymes. Inanother aspect, the enzyme composition comprises one or more (several)cellulolytic enzymes. In another aspect, the enzyme compositioncomprises one or more (several) xylan degrading enzymes and one or more(several) cellulolytic enzymes.

The one or more (several) xylan degrading enzymes are preferablyselected from the group consisting of a xylanase, an acetyxylanesterase, a feruloyl esterase, an arabinofuranosidase, a xylosidase, anda glucuronidase. The one or more (several) cellulolytic enzymes arepreferably selected from the group consisting of an endoglucanase, acellobiohydrolase, and a beta-glucosidase.

In another preferred aspect, the enzyme composition further or evenfurther comprises a polypeptide having cellulolytic enhancing activity(see, for example, WO 2005/074647, WO 2005/074656, and WO 2007/089290).In another aspect, the enzyme composition may further or even furthercomprise one or more (several) additional enzyme activities to improvethe degradation of the cellulose-containing material. Preferredadditional enzymes are hemicellulases (e.g., alpha-D-glucuronidases,alpha-L-arabinofuranosidases, endo-mannanases, beta-mannosidases,alpha-galactosidases, endo-alpha-L-arabinanases, beta-galactosidases),carbohydrate-esterases (e.g., acetylxylan esterases, acetyl-mannanesterases, ferulic acid esterases, coumaric acid esterases, glucuronoylesterases), pectinases, proteases, ligninolytic enzymes (e.g., laccases,manganese peroxidases, lignin peroxidases, H₂O₂-producing enzymes,oxidoreductases), expansins, swollenins, or mixtures thereof. In themethods of the present invention, the additional enzyme(s) can be addedprior to or during fermentation, e.g., during saccharification or duringor after propagation of the fermenting microorganism(s).

One or more (several) components of the enzyme composition may bewild-type proteins, recombinant proteins, or a combination of wild-typeproteins and recombinant proteins. For example, one or more (several)components may be native proteins of a cell, which is used as a hostcell to express recombinantly one or more (several) other components ofthe enzyme composition. One or more (several) components of the enzymecomposition may be produced as monocomponents, which are then combinedto form the enzyme composition. The enzyme composition may be acombination of multicomponent and monocomponent protein preparations.

The enzymes used in the methods of the present invention may be in anyform suitable for use in the processes described herein, such as, forexample, a crude fermentation broth with or without cells removed, asemi-purified or purified enzyme preparations, or a host cell as asource of the enzymes. The enzyme composition may be a dry powder orgranulate, a non-dusting granulate, a liquid, a stabilized liquid, or astabilized protected enzyme. Liquid enzyme preparations may, forinstance, be stabilized by adding stabilizers such as a sugar, a sugaralcohol or another polyol, and/or lactic acid or another organic acidaccording to established processes.

The optimum amounts of the enzymes and polypeptides having feruloylesterase activity depend on several factors including, but not limitedto, the mixture of component cellulolytic enzymes, the cellulosic orxylan-containing material, the concentration of the cellulosic orxylan-containing material, the pretreatment(s) of the cellulosic orxylan-containing material, temperature, time, pH, and inclusion offermenting organism (e.g., yeast for Simultaneous Saccharification andFermentation).

In a preferred aspect, an effective amount of cellulolytic enzyme(s)and/or xylan-degrading enzyme(s) to cellulosic or xylan-containingmaterial is about 0.5 to about 50 mg, preferably at about 0.5 to about40 mg, more preferably at about 0.5 to about 25 mg, more preferably atabout 0.75 to about 20 mg, more preferably at about 0.75 to about 15 mg,even more preferably at about 0.5 to about 10 mg, and most preferably atabout 2.5 to about 10 mg per g of cellulosic or xylan-containingmaterial.

In another preferred aspect, an effective amount of polypeptide(s)having feruloyl esterase activity to cellulosic or xylan-containingmaterial is about 0.01 to about 50.0 mg, preferably about 0.01 to about40 mg, more preferably about 0.01 to about 30 mg, more preferably about0.01 to about 20 mg, more preferably about 0.01 to about 10 mg, morepreferably about 0.01 to about 5 mg, more preferably at about 0.025 toabout 1.5 mg, more preferably at about 0.05 to about 1.25 mg, morepreferably at about 0.075 to about 1.25 mg, more preferably at about 0.1to about 1.25 mg, even more preferably at about 0.15 to about 1.25 mg,and most preferably at about 0.25 to about 1.0 mg per g of cellulosic orxylan-containing material.

In another preferred aspect, an effective amount of polypeptide(s)having feruloyl esterase activity to cellulolytic enzyme(s) and/orxylan-degrading enzyme(s) is about 0.005 to about 1.0 g, preferably atabout 0.01 to about 1.0 g, more preferably at about 0.15 to about 0.75g, more preferably at about 0.15 to about 0.5 g, more preferably atabout 0.1 to about 0.5 g, even more preferably at about 0.1 to about 0.5g, and most preferably at about 0.05 to about 0.2 g per g ofcellulolytic enzyme(s).

The enzymes can be derived or obtained from any suitable origin,including, bacterial, fungal, yeast, plant, or mammalian origin. Theterm “obtained” means herein that the enzyme may have been isolated froman organism that naturally produces the enzyme as a native enzyme. Theterm “obtained” also means herein that the enzyme may have been producedrecombinantly in a host organism employing methods described herein,wherein the recombinantly produced enzyme is either native or foreign tothe host organism or has a modified amino acid sequence, e.g., havingone or more (several) amino acids that are deleted, inserted and/orsubstituted, i.e., a recombinantly produced enzyme that is a mutantand/or a fragment of a native amino acid sequence or an enzyme producedby nucleic acid shuffling processes known in the art. Encompassed withinthe meaning of a native enzyme are natural variants and within themeaning of a foreign enzyme are variants obtained recombinantly, such asby site-directed mutagenesis or shuffling.

A polypeptide having cellulolytic enzyme activity or xylan degradingactivity may be a bacterial polypeptide. For example, the polypeptidemay be a gram positive bacterial polypeptide such as a Bacillus,Streptococcus, Streptomyces, Staphylococcus, Enterococcus,Lactobacillus, Lactococcus, Clostridium, Geobacillus, or Oceanobacilluspolypeptide having cellulolytic enzyme activity or xylan degradingactivity, or a Gram negative bacterial polypeptide such as an E. coli,Pseudomonas, Salmonella, Campylobacter, Helicobacter, Flavobacterium,Fusobacterium, Ilyobacter, Neisseria, or Ureaplasma polypeptide havingcellulolytic enzyme activity or xylan degrading activity.

In a preferred aspect, the polypeptide is a Bacillus alkalophilus,Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans,Bacillus clausii, Bacillus coagulans, Bacillus firmus, Bacillus lautus,Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacilluspumilus, Bacillus stearothermophilus, Bacillus subtilis, or Bacillusthuringiensis polypeptide having cellulolytic enzyme activity or xylandegrading activity.

In another preferred aspect, the polypeptide is a Streptococcusequisimilis, Streptococcus pyogenes, Streptococcus uberis, orStreptococcus equi subsp. Zooepidemicus polypeptide having cellulolyticenzyme activity or xylan degrading activity.

In another preferred aspect, the polypeptide is a Streptomycesachromogenes, Streptomyces avermitilis, Streptomyces coelicolor,Streptomyces griseus, or Streptomyces lividans polypeptide havingcellulolytic enzyme activity or xylan degrading activity.

The polypeptide having cellulolytic enzyme activity or xylan degradingactivity may also be a fungal polypeptide, and more preferably a yeastpolypeptide such as a Candida, Kluyveromyces, Pichia, Saccharomyces,Schizosaccharomyces, or Yarrowia polypeptide having cellulolytic enzymeactivity or xylan degrading activity; or more preferably a filamentousfungal polypeptide such as an Acremonium, Agaricus, Alternaria,Aspergillus, Aureobasidium, Botryospaeria, Ceriporiopsis, Chaetomidium,Chrysosporium, Claviceps, Cochliobolus, Coprinopsis, Coptotermes,Corynascus, Cryphonectria, Cryptococcus, Diplodia, Exidia, Filibasidium,Fusarium, Gibberella, Holomastigotoides, Humicola, Irpex, Lentinula,Leptospaeria, Magnaporthe, Melanocarpus, Meripilus, Mucor,Myceliophthora, Neocallimastix, Neurospora, Paecilomyces, Penicillium,Phanerochaete, Piromyces, Poitrasia, Pseudoplectania,Pseudotrichonympha, Rhizomucor, Schizophyllum, Scytalidium, Talaromyces,Thermoascus, Thielavia, Tolypocladium, Trichoderma, Trichophaea,Verticillium, Volvariella, or Xylaria polypeptide having cellulolyticenzyme activity or xylan degrading activity.

In a preferred aspect, the polypeptide is a Saccharomycescarlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus,Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomycesnorbensis, or Saccharomyces oviformis polypeptide having cellulolyticenzyme activity or xylan degrading activity.

In another preferred aspect, the polypeptide is an Acremoniumcellulolyticus, Aspergillus aculeatus, Aspergillus awamori, Aspergillusfumigatus, Aspergillus foetidus, Aspergillus japonicus, Aspergillusnidulans, Aspergillus niger, Aspergillus oryzae, Chrysosporiumkeratinophilum, Chrysosporium lucknowense, Chrysosporium tropicum,Chrysosporium merdarium, Chrysosporium inops, Chrysosporium pannicola,Chrysosporium queenslandicum, Chrysosporium zonatum, Fusariumbactridioides, Fusarium cerealis, Fusarium crookwellense, Fusariumculmorum, Fusarium graminearum, Fusarium graminum, Fusariumheterosporum, Fusarium negundi, Fusarium oxysporum, Fusariumreticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum,Fusarium sporotrichioides, Fusarium sulphureum, Fusarium torulosum,Fusarium trichothecioides, Fusarium venenatum, Humicola grisea, Humicolainsolens, Humicola lanuginosa, Irpex lacteus, Mucor miehei,Myceliophthora thermophila, Neurospora crassa, Penicillium funiculosum,Penicillium purpurogenum, Phanerochaete chrysosporium, Thielaviaachromatica, Thielavia albomyces, Thielavia albopilosa, Thielaviaaustraleinsis, Thielavia fimeti, Thielavia microspora, Thielaviaovispora, Thielavia peruviana, Thielavia spededonium, Thielavia setosa,Thielavia subthermophila, Thielavia terrestris, Trichoderma harzianum,Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei,Trichoderma viride, or Trichophaea saccata polypeptide havingcellulolytic enzyme activity or xylan degrading activity.

Chemically modified or protein engineered mutants of polypeptides havingcellulolytic enzyme activity or xylan degrading activity may also beused.

One or more (several) components of the enzyme composition may be arecombinant component, i.e., produced by cloning of a DNA sequenceencoding the single component and subsequent cell transformed with theDNA sequence and expressed in a host (see, for example, WO 91/17243 andWO 91/17244). The host is preferably a heterologous host (enzyme isforeign to host), but the host may under certain conditions also be ahomologous host (enzyme is native to host). Monocomponent cellulolyticproteins may also be prepared by purifying such a protein from afermentation broth.

Examples of commercial cellulolytic protein preparations suitable foruse in the present invention include, for example, CELLIC™ Ctec(Novozymes A/S), CELLUCLAST™ (Novozymes A/S), NOVOZYM™ 188 (NovozymesA/S), CELLUZYME™ (Novozymes A/S), CEREFLO™ (Novozymes A/S), andULTRAFLO™ (Novozymes A/S), ACCELERASE™ (Genencor Int.), LAMINEX™(Genencor Int.), SPEZYME™ CP (Genencor Int.), ROHAMENT™ 7069 W (RohmGmbH), FIBREZYME® LDI (Dyadic International, Inc.), FIBREZYME® LBR(Dyadic International, Inc.), or VISCOSTAR® 150L (Dyadic International,Inc.). The cellulase enzymes are added in amounts effective from about0.001 to about 5.0 wt % of solids, more preferably from about 0.025 toabout 4.0 wt % of solids, and most preferably from about 0.005 to about2.0 wt % of solids.

Examples of bacterial endoglucanases that can be used in the methods ofthe present invention, include, but are not limited to, an Acidothermuscellulolyticus endoglucanase (WO 91/05039; WO 93/15186; U.S. Pat. No.5,275,944; WO 96/02551; U.S. Pat. No. 5,536,655, WO 00/70031, WO05/093050); Thermobifida fusca endoglucanase III (WO 05/093050); andThermobifida fusca endoglucanase V (WO 05/093050).

Examples of fungal endoglucanases that can be used in the methods of thepresent invention, include, but are not limited to, a Trichoderma reeseiendoglucanase I (Penttila et al., 1986, Gene 45: 253-263; GENBANK™accession no. M15665); Trichoderma reesei endoglucanase II (Saloheimo,et al., 1988, Gene 63:11-22; GENBANK™ accession no. M19373); Trichodermareesei endoglucanase III (Okada et al., 1988, Appl. Environ. Microbiol.64: 555-563; GENBANK™ accession no. AB003694); Trichoderma reeseiendoglucanase IV (Saloheimo et al., 1997, Eur. J. Biochem. 249: 584-591;GENBANK™ accession no. Y11113); and Trichoderma reesei endoglucanase V(Saloheimo et al., 1994, Molecular Microbiology 13: 219-228; GENBANK™accession no. Z33381); Aspergillus aculeatus endoglucanase (Ooi et al.,1990, Nucleic Acids Research 18: 5884); Aspergillus kawachiiendoglucanase (Sakamoto et al., 1995, Current Genetics 27: 435-439);Erwinia carotovara endoglucanase (Saarilahti et al., 1990, Gene 90:9-14); Fusarium oxysporum endoglucanase (GENBANK™ accession no. L29381);Humicola grisea var. thermoidea endoglucanase (GENBANK™ accession no.AB003107); Melanocarpus albomyces endoglucanase (GENBANK™ accession no.MAL515703); Neurospora crassa endoglucanase (GENBANK™ accession no.XM_(—)324477); Humicola insolens endoglucanase V; Myceliophthorathermophila CBS 117.65 endoglucanase; basidiomycete CBS 495.95endoglucanase; basidiomycete CBS 494.95 endoglucanase; Thielaviaterrestris NRRL 8126 CEL6B endoglucanase; Thielavia terrestris NRRL 8126CEL6C endoglucanase); Thielavia terrestris NRRL 8126 CEL7Cendoglucanase; Thielavia terrestris NRRL 8126 CEL7E endoglucanase;Thielavia terrestris NRRL 8126 CEL7F endoglucanase; Cladorrhinumfoecundissimum ATCC 62373 CEL7A endoglucanase; and Trichoderma reeseistrain No. VTT-D-80133 endoglucanase (GENBANK™ accession no. M15665).

Examples of cellobiohydrolases useful in the methods of the presentinvention include, but are not limited to, Trichoderma reeseicellobiohydrolase I; Trichoderma reesei cellobiohydrolase II; Humicolainsolens cellobiohydrolase I, Myceliophthora thermophilacellobiohydrolase II, Thielavia terrestris cellobiohydrolase II (CEL6A),Chaetomium thermophilum cellobiohydrolase I, and Chaetomium thermophilumcellobiohydrolase II.

Examples of beta-glucosidases useful in the methods of the presentinvention include, but are not limited to, Aspergillus oryzaebeta-glucosidase; Aspergillus fumigatus beta-glucosidase; Penicilliumbrasilianum IBT 20888 beta-glucosidase; Aspergillus nigerbeta-glucosidase; and Aspergillus aculeatus beta-glucosidase.

The Aspergillus oryzae polypeptide having beta-glucosidase activity canbe obtained according to WO 2002/095014. The Aspergillus fumigatuspolypeptide having beta-glucosidase activity can be obtained accordingto WO 2005/047499. The Penicillium brasilianum polypeptide havingbeta-glucosidase activity can be obtained according to WO 2007/019442.The Aspergillus niger polypeptide having beta-glucosidase activity canbe obtained according to Dan et al., 2000, J. Biol. Chem. 275:4973-4980. The Aspergillus aculeatus polypeptide having beta-glucosidaseactivity can be obtained according to Kawaguchi et al., 1996, Gene 173:287-288.

The beta-glucosidase may be a fusion protein. In one aspect, thebeta-glucosidase is the Aspergillus oryzae beta-glucosidase variant BGfusion protein or the Aspergillus oryzae beta-glucosidase fusion proteinobtained according to WO 2008/057637.

Other endoglucanases, cellobiohydrolases, and beta-glucosidases aredisclosed in numerous Glycosyl Hydrolase families using theclassification according to Henrissat B., 1991, A classification ofglycosyl hydrolases based on amino-acid sequence similarities, Biochem.J. 280: 309-316, and Henrissat B., and Bairoch A., 1996, Updating thesequence-based classification of glycosyl hydrolases, Biochem. J. 316:695-696.

Other cellulolytic enzymes that may be used in the present invention aredescribed in EP 495,257, EP 531,315, EP 531,372, WO 89/09259, WO94/07998, WO 95/24471, WO 96/11262, WO 96/29397, WO 96/034108, WO97/14804, WO 98/08940, WO 98/012307, WO 98/13465, WO 98/015619, WO98/015633, WO 98/028411, WO 99/06574, WO 99/10481, WO 99/025846, WO99/025847, WO 99/031255, WO 2000/009707, WO 2002/050245, WO2002/0076792, WO 2002/101078, WO 2003/027306, WO 2003/052054, WO2003/052055, WO 2003/052056, WO 2003/052057, WO 2003/052118, WO2004/016760, WO 2004/043980, WO 2004/048592, WO 2005/001065, WO2005/028636, WO 2005/093050, WO 2005/093073, WO 2006/074005, WO2006/117432, WO 2007/071818, WO 2007/071820, WO 2008/008070, WO2008/008793, U.S. Pat. No. 4,435,307, U.S. Pat. No. 5,457,046, U.S. Pat.No. 5,648,263, U.S. Pat. No. 5,686,593, U.S. Pat. No. 5,691,178, U.S.Pat. No. 5,763,254, and U.S. Pat. No. 5,776,757.

In the methods of the present invention, any polypeptide havingcellulolytic enhancing activity can be used.

In a first aspect, the polypeptide having cellulolytic enhancingactivity comprises the following motifs:

[ILMV]-P-X(4,5)-G-X-Y-[ILMV]-X-R-X-[EQ]-X(4)-[HNQ] and[FW]-[TF]-K-[AIV],

wherein X is any amino acid, X(4,5) is any amino acid at 4 or 5contiguous positions, and X(4) is any amino acid at 4 contiguouspositions.

The polypeptide comprising the above-noted motifs may further comprise:

H-X(1,2)-G-P-X(3)-[YW]-[AILMV], [EQ]-X-Y-X(2)-C-X-[EHQN]-[FILV]-X-[ILV],or H-X(1,2)-G-P-X(3)-[YW]-[AILMV] and[EQ]-X-Y-X(2)-C-X-[EHQN]-[FILV]-X-[ILV],

wherein X is any amino acid, X(1,2) is any amino acid at 1 position or 2contiguous positions, X(3) is any amino acid at 3 contiguous positions,and X(2) is any amino acid at 2 contiguous positions. In the abovemotifs, the accepted IUPAC single letter amino acid abbreviation isemployed.

In a preferred aspect, the polypeptide having cellulolytic enhancingactivity further comprises H-X(1,2)-G-P-X(3)-[YW]-[AILMV]. In anotherpreferred aspect, the isolated polypeptide having cellulolytic enhancingactivity further comprises [EQ]-X-Y-X(2)-C-X-[EHQN]-[FILV]-X-[ILV]. Inanother preferred aspect, the polypeptide having cellulolytic enhancingactivity further comprises H-X(1,2)-G-P-X(3)-[YW]-[AILMV] and[EQ]-X-Y-X(2)-C-X-[EHQN]-[FILV]-X-[ILV].

In a second aspect, the polypeptide having cellulolytic enhancingactivity comprises the following motif:

[ILMV]-P-x(4,5)-G-x-Y-[ILMV]-x-R-x-[EQ]-x(3)-A-[HNQ],

wherein x is any amino acid, x(4,5) is any amino acid at 4 or 5contiguous positions, and x(3) is any amino acid at 3 contiguouspositions. In the above motif, the accepted IUPAC single letter aminoacid abbreviation is employed.

Examples of polypeptides having cellulolytic enhancing activity usefulin the methods of the present invention include, but are not limited to,polypeptides having cellulolytic enhancing activity from Thielaviaterrestris (WO 2005/074647); polypeptides having cellulolytic enhancingactivity from Thermoascus aurantiacus (WO 2005/074656); and polypeptideshaving cellulolytic enhancing activity from Trichoderma reesei (WO2007/089290).

Examples of commercial xylan degrading enzyme preparations suitable foruse in the present invention include, for example, SHEARZYME™ (NovozymesA/S), CELLIC™ Htec (Novozymes A/S), VISCOZYME® (Novozymes A/S),ULTRAFLO® (Novozymes A/S), PULPZYME® HC (Novozymes A/S), MULTIFECT®Xylanase (Genencor Int.), ECOPULP® TX-200A (AB Enzymes), HSP 6000Xylanase (DSM), DEPOL™ 333P (Biocatalysts Limit, Wales, UK), DEPOL™ 740L(Biocatalysts Limit, Wales, UK), and DEPOL™ 762P (Biocatalysts Limit,Wales, UK).

Examples of xylanases useful in the methods of the present inventioninclude, but are not limited to, Aspergillus aculeatus xylanase(GeneSeqP:AAR63790; WO 94/21785), Aspergillus fumigatus xylanases (WO2006/078256), and Thielavia terrestris NRRL 8126 xylanases (WO2009/079210).

Examples of beta-xylosidases useful in the methods of the presentinvention include, but are not limited to, Trichoderma reeseibeta-xylosidase (UniProtKB/TrEMBL accession number Q92458), Talaromycesemersonii (SwissProt accession number Q8X212), and Neurospora crassa(SwissProt accession number Q7SOW4).

Examples of acetylxylan esterases useful in the methods of the presentinvention include, but are not limited to, Hypocrea jecorina acetylxylanesterase (WO 2005/001036), Neurospora crassa acetylxylan esterase(UniProt accession number q7s259), Thielavia terrestris NRRL 8126acetylxylan esterase (WO 2009/042846), Chaetomium globosum acetylxylanesterase (Uniprot accession number Q2GWX4), Chaetomium gracileacetylxylan esterase (GeneSeqP accession number AAB82124), Phaeosphaerianodorum acetylxylan esterase (Uniprot accession number Q0UHJ1), andHumicola insolens DSM 1800 acetylxylan esterase (WO 2009/073709).

Examples of feruloyl esterases useful in the methods of the presentinvention include, but are not limited to, Humicola insolens DSM 1800feruloyl esterase (WO 2009/076122), Neurospora crassa feruloyl esterase(UniProt accession number Q9HGR3), and Neosartorya fischeri feruloylesterase (UniProt Accession number A1D9T4).

Examples of arabinofuranosidases useful in the methods of the presentinvention include, but are not limited to, Humicola insolens DSM 1800arabinofuranosidase (WO 2009/073383) and Aspergillus nigerarabinofuranosidase (GeneSeqP accession number AAR94170).

Examples of alpha-glucuronidases useful in the methods of the presentinvention include, but are not limited to, Aspergillus clavatusalpha-glucuronidase (UniProt accession number alcc12), Trichodermareesei alpha-glucuronidase (Uniprot accession number Q99024),Talaromyces emersonii alpha-glucuronidase (UniProt accession numberQ8X211), Aspergillus niger alpha-glucuronidase (Uniprot accession numberQ96WX9), Aspergillus terreus alpha-glucuronidase (SwissProt accessionnumber Q0CJP9), and Aspergillus fumigatus alpha-glucuronidase (SwissProtaccession number Q4WW45).

The enzymes and proteins used in the methods of the present inventionmay be produced by fermentation of the above-noted microbial strains ona nutrient medium containing suitable carbon and nitrogen sources andinorganic salts, using procedures known in the art (see, e.g., Bennett,J. W. and LaSure, L. (eds.), More Gene Manipulations in Fungi, AcademicPress, CA, 1991). Suitable media are available from commercial suppliersor may be prepared according to published compositions (e.g., incatalogues of the American Type Culture Collection). Temperature rangesand other conditions suitable for growth and enzyme production are knownin the art (see, e.g., Bailey, J. E., and Ollis, D. F., BiochemicalEngineering Fundamentals, McGraw-Hill Book Company, NY, 1986).

The fermentation can be any method of cultivation of a cell resulting inthe expression or isolation of an enzyme. Fermentation may, therefore,be understood as comprising shake flask cultivation, or small- orlarge-scale fermentation (including continuous, batch, fed-batch, orsolid state fermentations) in laboratory or industrial fermentorsperformed in a suitable medium and under conditions allowing the enzymeto be expressed or isolated. The resulting enzymes produced by themethods described above may be recovered from the fermentation mediumand purified by conventional procedures.

Fermentation. The fermentable sugars obtained from the pretreated andhydrolyzed cellulosic or xylan-containing material can be fermented byone or more (several) fermenting microorganisms capable of fermentingthe sugars directly or indirectly into a desired fermentation product.“Fermentation” or “fermentation process” refers to any fermentationprocess or any process comprising a fermentation step. Fermentationprocesses also include fermentation processes used in the consumablealcohol industry (e.g., beer and wine), dairy industry (e.g., fermenteddairy products), leather industry, and tobacco industry. Thefermentation conditions depend on the desired fermentation product andfermenting organism and can easily be determined by one skilled in theart.

In the fermentation step, sugars, released from cellulosic orxylan-containing material as a result of the pretreatment and enzymatichydrolysis steps, are fermented to a product, e.g., ethanol, by afermenting organism, such as yeast. Hydrolysis (saccharification) andfermentation can be separate or simultaneous, as described herein.

Any suitable hydrolyzed cellulosic or xylan-containing material can beused in the fermentation step in practicing the present invention. Thematerial is generally selected based on the desired fermentationproduct, i.e., the substance to be obtained from the fermentation, andthe process employed, as is well known in the art.

The term “fermentation medium” is understood herein to refer to a mediumbefore the fermenting microorganism(s) is(are) added, such as, a mediumresulting from a saccharification process, as well as a medium used in asimultaneous saccharification and fermentation process (SSF).

“Fermenting microorganism” refers to any microorganism, includingbacterial and fungal organisms, suitable for use in a desiredfermentation process to produce a fermentation product. The fermentingorganism can be C₆ and/or C₅ fermenting organisms, or a combinationthereof. Both C₆ and C₅ fermenting organisms are well known in the art.Suitable fermenting microorganisms are able to ferment, i.e., convert,sugars, such as glucose, xylose, xylulose, arabinose, maltose, mannose,galactose, or oligosaccharides, directly or indirectly into the desiredfermentation product.

Examples of bacterial and fungal fermenting organisms producing ethanolare described by Lin et al., 2006, Appl. Microbiol. Biotechnol. 69:627-642.

Examples of fermenting microorganisms that can ferment C6 sugars includebacterial and fungal organisms, such as yeast. Preferred yeast includesstrains of the Saccharomyces spp., preferably Saccharomyces cerevisiae.

Examples of fermenting organisms that can ferment C5 sugars includebacterial and fungal organisms, such as yeast. Preferred C5 fermentingyeast include strains of Pichia, preferably Pichia stipitis, such asPichia stipitis CBS 5773; strains of Candida, preferably Candidaboidinii, Candida brassicae, Candida sheatae, Candida diddensii, Candidapseudotropicalis, or Candida utilis.

Other fermenting organisms include strains of Zymomonas, such asZymomonas mobilis; Hansenula, such as Hansenula anomala; Kluyveromyces,such as K. fragilis; Schizosaccharomyces, such as S. pombe; and E. coli,especially E. coli strains that have been genetically modified toimprove the yield of ethanol.

In a preferred aspect, the yeast is a Saccharomyces spp. In a morepreferred aspect, the yeast is Saccharomyces cerevisiae. In another morepreferred aspect, the yeast is Saccharomyces distaticus. In another morepreferred aspect, the yeast is Saccharomyces uvarum. In anotherpreferred aspect, the yeast is a Kluyveromyces. In another morepreferred aspect, the yeast is Kluyveromyces marxianus. In another morepreferred aspect, the yeast is Kluyveromyces fragilis. In anotherpreferred aspect, the yeast is a Candida. In another more preferredaspect, the yeast is Candida boidinii. In another more preferred aspect,the yeast is Candida brassicae. In another more preferred aspect, theyeast is Candida diddensii. In another more preferred aspect, the yeastis Candida pseudotropicalis. In another more preferred aspect, the yeastis Candida utilis. In another preferred aspect, the yeast is aClavispora. In another more preferred aspect, the yeast is Clavisporalusitaniae. In another more preferred aspect, the yeast is Clavisporaopuntiae. In another preferred aspect, the yeast is a Pachysolen. Inanother more preferred aspect, the yeast is Pachysolen tannophilus. Inanother preferred aspect, the yeast is a Pichia. In another morepreferred aspect, the yeast is a Pichia stipitis. In another preferredaspect, the yeast is a Bretannomyces. In another more preferred aspect,the yeast is Bretannomyces clausenii (Philippidis, G. P., 1996,Cellulose bioconversion technology, in Handbook on Bioethanol:Production and Utilization, Wyman, C. E., ed., Taylor & Francis,Washington, D.C., 179-212).

Bacteria that can efficiently ferment hexose and pentose to ethanolinclude, for example, Zymomonas mobilis and Clostridium thermocellum(Philippidis, 1996, supra).

In a preferred aspect, the bacterium is a Zymomonas. In a more preferredaspect, the bacterium is Zymomonas mobilis. In another preferred aspect,the bacterium is a Clostridium. In another more preferred aspect, thebacterium is Clostridium thermocellum.

Commercially available yeast suitable for ethanol production includes,e.g., ETHANOL RED™ yeast (available from Fermentis/Lesaffre, USA), FALI™(available from Fleischmann's Yeast, USA), SUPERSTART™ and THERMOSACC™fresh yeast (available from Ethanol Technology, WI, USA), BIOFERM™ AFTand XR (available from NABC—North American Bioproducts Corporation, GA,USA), GERT STRAND™ (available from Gert Strand AB, Sweden), and FERMIOL™(available from DSM Specialties).

In a preferred aspect, the fermenting microorganism has been geneticallymodified to provide the ability to ferment pentose sugars, such asxylose utilizing, arabinose utilizing, and xylose and arabinoseco-utilizing microorganisms.

The cloning of heterologous genes into various fermenting microorganismshas led to the construction of organisms capable of converting hexosesand pentoses to ethanol (cofermentation) (Chen and Ho, 1993, Cloning andimproving the expression of Pichia stipitis xylose reductase gene inSaccharomyces cerevisiae, Appl. Biochem. Biotechnol. 39-40: 135-147; Hoet al., 1998, Genetically engineered Saccharomyces yeast capable ofeffectively cofermenting glucose and xylose, Appl. Environ. Microbiol.64: 1852-1859; Kotter and Ciriacy, 1993, Xylose fermentation bySaccharomyces cerevisiae, Appl. Microbiol. Biotechnol. 38: 776-783;Walfridsson et al., 1995, Xylose-metabolizing Saccharomyces cerevisiaestrains overexpressing the TKL1 and TAL1 genes encoding the pentosephosphate pathway enzymes transketolase and transaldolase, Appl.Environ. Microbiol. 61: 4184-4190; Kuyper et al., 2004, Minimalmetabolic engineering of Saccharomyces cerevisiae for efficientanaerobic xylose fermentation: a proof of principle, FEMS Yeast Research4: 655-664; Beall et al., 1991, Parametric studies of ethanol productionfrom xylose and other sugars by recombinant Escherichia coli, Biotech.Bioeng. 38: 296-303; Ingram et al., 1998, Metabolic engineering ofbacteria for ethanol production, Biotechnol. Bioeng. 58: 204-214; Zhanget al., 1995, Metabolic engineering of a pentose metabolism pathway inethanologenic Zymomonas mobilis, Science 267: 240-243; Deanda et al.,1996, Development of an arabinose-fermenting Zymomonas mobilis strain bymetabolic pathway engineering, Appl. Environ. Microbiol. 62: 4465-4470;WO 2003/062430, xylose isomerase).

In a preferred aspect, the genetically modified fermenting microorganismis Saccharomyces cerevisiae. In another preferred aspect, thegenetically modified fermenting microorganism is Zymomonas mobilis. Inanother preferred aspect, the genetically modified fermentingmicroorganism is Escherichia coli. In another preferred aspect, thegenetically modified fermenting microorganism is Klebsiella oxytoca. Inanother preferred aspect, the genetically modified fermentingmicroorganism is Kluyveromyces sp.

It is well known in the art that the organisms described above can alsobe used to produce other substances, as described herein.

The fermenting microorganism is typically added to the degradedlignocellulose or hydrolysate and the fermentation is performed forabout 8 to about 96 hours, such as about 24 to about 60 hours. Thetemperature is typically between about 26° C. to about 60° C., inparticular about 32° C. or 50° C., and at about pH 3 to about pH 8, suchas around pH 4-5, 6, or 7.

In a preferred aspect, the yeast and/or another microorganism is appliedto the degraded cellulosic or xylan-containing material and thefermentation is performed for about 12 to about 96 hours, such astypically 24-60 hours. In a preferred aspect, the temperature ispreferably between about 20° C. to about 60° C., more preferably about25° C. to about 50° C., and most preferably about 32° C. to about 50°C., in particular about 32° C. or 50° C., and the pH is generally fromabout pH 3 to about pH 7, preferably around pH 4-7. However, somefermenting organisms, e.g., bacteria, have higher fermentationtemperature optima. Yeast or another microorganism is preferably appliedin amounts of approximately 10⁵ to 10¹², preferably from approximately10⁷ to 10¹⁰, especially approximately 2×10⁸ viable cell count per ml offermentation broth. Further guidance in respect of using yeast forfermentation can be found in, e.g., “The Alcohol Textbook” (Editors K.Jacques, T. P. Lyons and D. R. Kelsall, Nottingham University Press,United Kingdom 1999), which is hereby incorporated by reference.

For ethanol production, following the fermentation the fermented slurryis distilled to extract the ethanol. The ethanol obtained according tothe methods of the invention can be used as, e.g., fuel ethanol,drinking ethanol, i.e., potable neutral spirits, or industrial ethanol.

A fermentation stimulator can be used in combination with any of theprocesses described herein to further improve the fermentation process,and in particular, the performance of the fermenting microorganism, suchas, rate enhancement and ethanol yield. A “fermentation stimulator”refers to stimulators for growth of the fermenting microorganisms, inparticular, yeast. Preferred fermentation stimulators for growth includevitamins and minerals. Examples of vitamins include multivitamins,biotin, pantothenate, nicotinic acid, meso-inositol, thiamine,pyridoxine, para-aminobenzoic acid, folic acid, riboflavin, and VitaminsA, B, C, D, and E. See, for example, Alfenore et al., Improving ethanolproduction and viability of Saccharomyces cerevisiae by a vitaminfeeding strategy during fed-batch process, Springer-Verlag (2002), whichis hereby incorporated by reference. Examples of minerals includeminerals and mineral salts that can supply nutrients comprising P, K,Mg, S, Ca, Fe, Zn, Mn, and Cu.

Fermentation products: A fermentation product can be any substancederived from the fermentation. The fermentation product can be, withoutlimitation, an alcohol (e.g., arabinitol, butanol, ethanol, glycerol,methanol, 1,3-propanediol, sorbitol, and xylitol); an organic acid(e.g., acetic acid, acetonic acid, adipic acid, ascorbic acid, citricacid, 2,5-diketo-D-gluconic acid, formic acid, fumaric acid, glucaricacid, gluconic acid, glucuronic acid, glutaric acid, 3-hydroxypropionicacid, itaconic acid, lactic acid, malic acid, malonic acid, oxalic acid,propionic acid, succinic acid, and xylonic acid); a ketone (e.g.,acetone); an amino acid (e.g., aspartic acid, glutamic acid, glycine,lysine, serine, and threonine); and a gas (e.g., methane, hydrogen (H₂),carbon dioxide (CO₂), and carbon monoxide (CO)). The fermentationproduct can also be protein as a high value product.

In a preferred aspect, the fermentation product is an alcohol. It willbe understood that the term “alcohol” encompasses a substance thatcontains one or more hydroxyl moieties. In a more preferred aspect, thealcohol is arabinitol. In another more preferred aspect, the alcohol isbutanol. In another more preferred aspect, the alcohol is ethanol. Inanother more preferred aspect, the alcohol is glycerol. In another morepreferred aspect, the alcohol is methanol. In another more preferredaspect, the alcohol is 1,3-propanediol. In another more preferredaspect, the alcohol is sorbitol. In another more preferred aspect, thealcohol is xylitol. See, for example, Gong, C. S., Cao, N. J., Du, J.,and Tsao, G. T., 1999, Ethanol production from renewable resources, inAdvances in Biochemical Engineering/Biotechnology, Scheper, T., ed.,Springer-Verlag Berlin Heidelberg, Germany, 65: 207-241; Silveira, M.M., and Jonas, R., 2002, The biotechnological production of sorbitol,Appl. Microbiol. Biotechnol. 59: 400-408; Nigam, P., and Singh, D.,1995, Processes for fermentative production of xylitol—a sugarsubstitute, Process Biochemistry 30 (2): 117-124; Ezeji, T. C., Qureshi,N. and Blaschek, H. P., 2003, Production of acetone, butanol and ethanolby Clostridium beijerinckii BA101 and in situ recovery by gas stripping,World Journal of Microbiology and Biotechnology 19 (6): 595-603.

In another preferred aspect, the fermentation product is an organicacid. In another more preferred aspect, the organic acid is acetic acid.In another more preferred aspect, the organic acid is acetonic acid. Inanother more preferred aspect, the organic acid is adipic acid. Inanother more preferred aspect, the organic acid is ascorbic acid. Inanother more preferred aspect, the organic acid is citric acid. Inanother more preferred aspect, the organic acid is 2,5-diketo-D-gluconicacid. In another more preferred aspect, the organic acid is formic acid.In another more preferred aspect, the organic acid is fumaric acid. Inanother more preferred aspect, the organic acid is glucaric acid. Inanother more preferred aspect, the organic acid is gluconic acid. Inanother more preferred aspect, the organic acid is glucuronic acid. Inanother more preferred aspect, the organic acid is glutaric acid. Inanother preferred aspect, the organic acid is 3-hydroxypropionic acid.In another more preferred aspect, the organic acid is itaconic acid. Inanother more preferred aspect, the organic acid is lactic acid. Inanother more preferred aspect, the organic acid is malic acid. Inanother more preferred aspect, the organic acid is malonic acid. Inanother more preferred aspect, the organic acid is oxalic acid. Inanother more preferred aspect, the organic acid is propionic acid. Inanother more preferred aspect, the organic acid is succinic acid. Inanother more preferred aspect, the organic acid is xylonic acid. See,for example, Chen, R., and Lee, Y. Y., 1997, Membrane-mediatedextractive fermentation for lactic acid production from cellulosicbiomass, Appl. Biochem. Biotechnol. 63-65: 435-448.

In another preferred aspect, the fermentation product is a ketone. Itwill be understood that the term “ketone” encompasses a substance thatcontains one or more ketone moieties. In another more preferred aspect,the ketone is acetone. See, for example, Qureshi and Blaschek, 2003,supra.

In another preferred aspect, the fermentation product is an amino acid.In another more preferred aspect, the organic acid is aspartic acid. Inanother more preferred aspect, the amino acid is glutamic acid. Inanother more preferred aspect, the amino acid is glycine. In anothermore preferred aspect, the amino acid is lysine. In another morepreferred aspect, the amino acid is serine. In another more preferredaspect, the amino acid is threonine. See, for example, Richard, A., andMargaritis, A., 2004, Empirical modeling of batch fermentation kineticsfor poly(glutamic acid) production and other microbial biopolymers,Biotechnology and Bioengineering 87 (4): 501-515.

In another preferred aspect, the fermentation product is a gas. Inanother more preferred aspect, the gas is methane. In another morepreferred aspect, the gas is H₂. In another more preferred aspect, thegas is CO₂. In another more preferred aspect, the gas is CO. See, forexample, Kataoka, N., A. Miya, and K. Kiriyama, 1997, Studies onhydrogen production by continuous culture system of hydrogen-producinganaerobic bacteria, Water Science and Technology 36 (6-7): 41-47; andGunaseelan V. N. in Biomass and Bioenergy, Vol. 13 (1-2), pp. 83-114,1997, Anaerobic digestion of biomass for methane production: A review.

Recovery. The fermentation product(s) can be optionally recovered fromthe fermentation medium using any method known in the art including, butnot limited to, chromatography, electrophoretic procedures, differentialsolubility, distillation, or extraction. For example, alcohol isseparated from the fermented cellulosic or xylan-containing material andpurified by conventional methods of distillation. Ethanol with a purityof up to about 96 vol. % can be obtained, which can be used as, forexample, fuel ethanol, drinking ethanol, i.e., potable neutral spirits,or industrial ethanol.

Other Uses

The polypeptides of the present invention may also be used with limitedactivity of other xylanolytic enzymes to degrade xylans for productionof oligosaccharides. The oligosaccharides may be used as bulking agents,like arabinoxylan oligosaccharides released from cereal cell wallmaterial, or more or less purified arabinoxylans from cereals.

The polypeptides of the present invention may also be used incombination with other xylanolytic enzymes to degrade xylans to xyloseand other monosaccharides (U.S. Pat. No. 5,658,765). The released xylosemay be converted to other compounds.

The polypeptides of the present invention may be used together withother enzymes like glucanases to improve the extraction of oil fromoil-rich plant material, like corn-oil from corn-embryos.

The polypeptides of the present invention may also be used in baking toimprove the development, elasticity, and/or stability of dough and/orthe volume, crumb structure, and/or anti-staling properties of the bakedproduct (see U.S. Pat. No. 5,693,518). The polypeptides may also be usedfor the preparation of dough or baked products prepared from any type offlour or meal (e.g., based on wheat, rye, barley, oat, or maize). Thebaked products produced with a polypeptide of the present inventioninclude bread, rolls, baguettes and the like. For baking purposes apolypeptide of the present invention may be used as the only or majorenzymatic activity, or may be used in combination with other enzymessuch as a xylanase, a lipase, an amylase, an oxidase (e.g., glucoseoxidase, peroxidase), a laccase and/or a protease.

The polypeptides of the present invention may also be used formodification of animal feed and may exert their effect either in vitro(by modifying components of the feed) or in vivo to improve feeddigestibility and increase the efficiency of its utilization (U.S. Pat.No. 6,245,546). The polypeptides may be added to animal feedcompositions containing high amounts of arabinoxylans andglucuronoxylans, e.g., feed containing cereals such as barley, wheat,rye, oats, or maize. When added to feed the polypeptide will improve thein vivo break-down of plant cell wall material partly due to a reductionof intestinal viscosity (Bedford et al., 1993, Proceedings of the 1stSymposium on Enzymes in Animal Nutrition, pp. 73-77), whereby improvedutilization of the plant nutrients by the animal is achieved. Thereby,the growth rate and/or feed conversion ratio (i.e., the weight ofingested feed relative to weight gain) of the animal is improved.

The polypeptides of the present invention may also be used in the paperand pulp industry, inter alia, in bleaching processes to enhance thebrightness of bleached pulps whereby the amount of chlorine used in thebleaching stages is reduced, and to increase the freeness of pulps inthe recycled paper process (Eriksson, 1990, Wood Science and Technology24: 79-101; Paice et al., 1988, Biotechnol. and Bioeng. 32: 235-239, andPommier et al., 1989, Tappi Journal 187-191). The treatment oflignocellulosic pulp may be performed, for example, as described in U.S.Pat. No. 5,658,765, WO 93/08275, WO 91/02839, and WO 92/03608.

The polypeptides of the present invention may also be used in beerbrewing, in particular to improve the filterability of wort containing,for example, barley and/or sorghum malt (WO 2002/24926). Thepolypeptides may be used in the same manner as pentosanasesconventionally used for brewing, e.g., as described by Viëtor et al.,1993, J. Inst. Brew. 99: 243-248; and EP 227159. Furthermore, thepolypeptides may be used for treatment of brewers spent grain, i.e.,residuals from beer wort production containing barley or malted barleyor other cereals, so as to improve the utilization of the residuals for,e.g., animal feed.

The polypeptides of the present invention may be used for separation ofcomponents of plant cell materials, in particular of cereal componentssuch as wheat components. Of particular interest is the separation ofwheat into gluten and starch, i.e., components of considerablecommercial interest. The separation process may be performed by use ofmethods known in the art, such as the so-called batter process (or wetmilling process) performed as a hydroclone or a decanter process. In thebatter process, the starting material is a dilute pumpable dispersion ofthe plant material such as wheat to be subjected to separation. In awheat separation process the dispersion is made normally from wheatflour and water.

The polypeptides of the invention may also be used in the preparation offruit or vegetable juice in order to increase yield (see, for example,U.S. Pat. No. 6,228,630).

The polypeptides of the present invention may also be used as acomponent of an enzymatic scouring system for textiles (see, forexample, U.S. Pat. No. 6,258,590).

The polypeptides of the present invention may also be used in laundrydetergent applications in combination with other enzyme functionalities(see, for example, U.S. Pat. No. 5,696,068).

Signal Peptide

The present invention also relates to an isolated polynucleotideencoding a signal peptide comprising or consisting of amino acids 1 to25 of SEQ ID NO: 2.

The present invention also relates to nucleic acid constructs comprisinga gene encoding a protein, wherein the gene is operably linked to one orboth of the isolated polynucleotide encoding the signal peptide and theisolated polynucleotide encoding the propeptide, wherein the gene isforeign to the polynucleotides encoding the signal peptide andpropeptide.

In a preferred aspect, the isolated polynucleotide encoding a signalpeptide comprises or consists of nucleotides 1 to 75 of SEQ ID NO: 1.

The present invention also relates to recombinant expression vectors andrecombinant host cells comprising such nucleic acid constructs.

The present invention also relates to methods of producing a protein,comprising: (a) cultivating a recombinant host cell comprising a geneencoding a protein operably linked to the such polynucleotides encodinga signal peptide, a propeptide, or a signal peptide and a propeptide,wherein the gene is foreign to the polynucleotide under conditionsconducive for production of the protein; and (b) recovering the protein;and (b) recovering the protein.

The protein may be native or heterologous to a host cell. The term“protein” is not meant herein to refer to a specific length of theencoded product and, therefore, encompasses peptides, oligopeptides, andproteins. The term “protein” also encompasses two or more polypeptidescombined to form the encoded product. The proteins also include hybridpolypeptides that comprise a combination of partial or completepolypeptide sequences obtained from at least two different proteinswherein one or more (several) may be heterologous or native to the hostcell. Proteins further include naturally occurring allelic andengineered variations of the above mentioned proteins and hybridproteins.

Preferably, the protein is a hormone or variant thereof, enzyme,receptor or portion thereof, antibody or portion thereof, or reporter.In a more preferred aspect, the protein is an oxidoreductase,transferase, hydrolase, lyase, isomerase, or ligase. In an even morepreferred aspect, the protein is an aminopeptidase, amylase,carbohydrase, carboxypeptidase, catalase, cellulase, chitinase,cutinase, cyclodextrin glycosyltransferase, deoxyribonuclease, esterase,alpha-galactosidase, beta-galactosidase, glucoamylase,alpha-glucosidase, beta-glucosidase, invertase, laccase, another lipase,mannosidase, mutanase, oxidase, pectinolytic enzyme, peroxidase,phytase, polyphenoloxidase, proteolytic enzyme, ribonuclease,transglutaminase or xylanase.

The gene may be obtained from any prokaryotic, eukaryotic, or othersource.

The present invention is further described by the following examplesthat should not be construed as limiting the scope of the invention.

EXAMPLES Materials

Chemicals used as buffers and substrates were commercial products of atleast reagent grade.

Strains

Thielavia terrestris NRRL 8126 was used as the source of a gene encodinga polypeptide having feruloyl esterase activity.

Media

LB medium was composed of 10 g of tryptone, 5 g of yeast extract, 5 g ofNaCl, and deionized water to 1 liter.

YPG medium was composed of 2% glucose, 10 g of yeast extract, 20 g ofBacto peptone, and deionized water to 1 liter.

PDA plates were composed of 39 g of potato dextrose agar and deionizedwater to 1 liter.

2XYT+ agar plates were composed of 16 g of tryptone, 10 g of yeastextract, 5 g of NaCl, 15 g of bacto agar, and 100 mg of ampicillin, anddeionized water to 1 liter.

Determination of Feruloyl Esterase Activity

Feruloyl esterase activity is determined using p-nitrophenylferulate assubstrate. The enzyme preparation is diluted to provide less than 15%conversion of p-nitrophenylferulate by making an initial dilution in a1.5 ml microcentrifuge tube with 50 mM sodium acetate pH 5.0 followed by2-fold serial dilutions with 50 mM sodium acetate pH 5.0. Then 100 μlaliquots of the diluted enzyme are transferred to wells of a 96-wellplate.

A p-nitrophenylferulate stock solution is made by dissolvingp-nitrophenylacetate in dimethylsulfoxide (DMSO) to constitute a 0.1 Msolution. Before assay, a sample of the stock solution is diluted100-fold in 50 mM sodium acetate pH 5.0 to make a 1 mM solution. A 100μl volume of 1 mM p-nitrophenylferulate is mixed with each dilution ofthe enzyme and then incubated at 25° C. for 10 minutes. Substrate alone,enzyme alone, and buffer alone are run as controls. p-Nitrophenolstandard solutions of 0.25, 0.2, 0.1, 0.05, and 0.02 mM are prepared bydiluting a 10 mM stock solution in 50 mM sodium acetate pH 5.0. At 10minutes, 50 μl of 1.0 M Tris-HCl pH 8.0 buffer is added to each well(including samples, substrate control, enzyme control, reagent control,and standards), mixed, and the absorbance at 405 nm immediately measuredon a SPECTRAMAX™ 340 PC plate reader (Molecular Devices, Sunnyvale,Calif., USA). One unit of feruloyl esterase activity is defined as theamount of enzyme capable of releasing 1 μmole of p-nitrophenolate anionper minute at pH 5, 25° C.

Example 1 Identification of a Feruloyl Esterase Gene in the GenomicSequence of Thielavia terrestris

A low redundancy draft sequence of the Thielavia terrestris NRRL 8126genome was generated by the Joint Genome Center (JGI), Walnut Creek,Calif., USA, using the whole genome shotgun method according to Martinezet al., 2008, Nature Biotechnol. 26: 553-560. Shotgun sequencing reads(approximately 18307) were assembled into contigs using the Phrapassembler (Ewing and Green, 1998, Genome Res. 8: 186-194). A tblastnsearch (Altschul et al., 1997, Nucleic Acids Res. 25: 3389-3402) of theassembled contigs was carried out using as query a feruloyl esteraseprotein sequence from Humicola insolens (SEQ ID NO: 3). A translatedamino acid sequence of 86 amino acids with greater than 76% identity tothe query sequence was identified. The partial sequence was searchedagainst public databases using blastp with BLOSUM62 as matrix and gappenalties set to 11 (Altschul et al., 1997, Nucleic Acids Res. 25:3389-3402) and was found to be 76% identical to a tannase fromAspergillus oryzae (UniProt accession number P78581).

Example 2 Identification of a Full-Length Feruloyl Esterase Gene in theGenomic Sequence of Thielavia terrestris

A low redundancy (approximately 4.5× coverage) draft sequence of theThielavia terrestris NRRL 8126 genome was generated by JGI, using thewhole genome shotgun method described by Martinez et al., 2008, supra.Shotgun sequencing reads (approximately 266822) were assembled intocontigs using the JAZZ assembler (Shapiro, H. 2005. “Outline of theassembly process: Jazz, the JGI in-house assembler” LBNL PaperLBNL-58236. Lawrence Berkeley National Laboratory, Berkeley, Calif.,USA), and gene models were derived from the assembled contigs using thePedantPro software suite (Biomax Informatics AG, Lochhamer Str. 9,D-82152 Martinsried, Germany).

The full-length amino acid sequence from Aspergillus oryzae tannase(UniProt accession number P78581) was used to search against theThielavia terrestris gene models using tblastn with BLOSUM62 as thematrix and gap penalties set to 11 (Altschul et al., 1997, supra). Theresults of the tblastn search identified a 558 bp section of the genemodels having 72% identity to the Aspergillus oryzae tannase sequence.

Example 3 Thielavia terrestris NRRL 8126 Genomic DNA Extraction

Thielavia terrestris NRRL 8126 was grown on PDA plates at 45° C. toconfluence. Three 4 mm² squares were cut from the PDA plates, inoculatedinto 25 ml of YP medium containing 2% glucose in a baffled 125 ml shakeflask, and incubated at 41° C. for 2 days with shaking at 200 rpm.Mycelia were harvested by filtration using MIRACLOTH® (Calbiochem, LaJolla, Calif., USA), washed twice in deionized water, and frozen underliquid nitrogen. Frozen mycelia were ground, by mortar and pestle, to afine powder, and total DNA was isolated using a DNEASY® Plant Maxi Kit(QIAGEN Inc., Valencia, Calif., USA).

Example 4 Cloning of the Thielavia terrestris Feruloyl Esterase Gene andConstruction of an Aspergillus niger Expression Vector

Two synthetic oligonucleotide primers (shown below) were designed basedon the gene model described in Example 2. These primers were used to PCRamplify the Thielavia terrestris feruloyl esterase gene from the genomicDNA prepared in Example 3. An IN-FUSION™ Cloning Kit (BD Biosciences,Palo Alto, Calif., USA) was used to clone the fragment directly into theexpression vector pBM120a (WO 2006/078256).

TtFaeCNcoTagV2F: (SEQ ID NO: 4) 5′-ACACAACTGGCCATGGCCGCCTTCGCCAAGCT-3′PacTagTtFaeCsca7R: (SEQ ID NO: 5)5′-CAGTCACCTCTAGTTATTAGTACACAGGCACCTTAA-3′

Bold letters represent coding sequence. The remaining sequence ishomologous to the insertion sites of pBM120a.

Fifty picomoles of each of the primers above were used in anamplification reaction composed of 95.4 ng of Thielavia terrestrisgenomic DNA, 1× EXPAND™ High Fidelity PCR buffer (Roche Applied Science,Indianapolis, Ind., USA) with MgCl₂, 0.25 mM each of dATP, dTTP, dGTP,and dCTP, and 2.6 units of EXPAND™ Enzyme Mix (Roche Applied Science,Indianapolis, Ind., USA) in a final volume of 50 μl. The amplificationwas performed using an EPPENDORF® MASTERCYCLER® 5333 (EppendorfScientific, Inc., Westbury, N.Y., USA) programmed for 1 cycle at 94° C.for 2 minute; 30 cycles each at 94° C. for 15 seconds, 61.4° C. for 30seconds, and 72° C. for 1 minute; and a final elongation at 72° C. for 7minutes. The heat block then went to a 4° C. soak cycle.

The reaction products were isolated by 0.7% agarose gel electrophoresisin TBE buffer (10.8 g of Tris base, 5.5 g of boric acid and 4 ml of 0.5M EDTA pH 8.0 per liter). A PCR product band of approximately 1.8 kb wasexcised from the gel and purified using a QIAQUICK® Gel Extraction Kit(QIAGEN Inc., Valencia, Calif., USA) according to the manufacturer'sinstructions.

Plasmid pBM120a was digested with Nco I and Pac I, isolated by 1.0%agarose gel electrophoresis in TBE buffer, and purified using aQIAQUICK® Gel Extraction Kit according to the manufacturer'sinstructions.

The gene fragment and the digested vector were ligated together using anIN-FUSION™ Cloning Kit resulting in pDFng106 (FIG. 2) in whichtranscription of the feruloyl esterase gene was under the control of ahybrid of promoters from the genes for Aspergillus niger neutralalpha-amylase and Aspergillus nidulans triose phosphate isomerase(NA2-tpi promoter). The ligation reaction (20 μl) was composed of 1×IN-FUSION™ Buffer (BD Biosciences, Palo Alto, Calif., USA), 1× BSA (BDBiosciences, Palo Alto, Calif., USA), 1 μl of IN-FUSION™ enzyme (diluted1:10) (BD Biosciences, Palo Alto, Calif., USA), 132 ng of pBM120adigested with Nco I and Pac I, and 100 ng of the purified Thielaviaterrestris PCR product. The reaction was incubated at room temperaturefor 30 minutes. Two μl of the reaction were used to transform E. coliXL10 SOLOPACK® Gold Supercompetent cells (Stratagene, La Jolla, Calif.,USA) according to the maufacturer's instructions. An E. colitransformant containing pDFng106 was obtained and plasmid DNA wasprepared using a BIOROBOT® 9600 (QIAGEN Inc., Valencia, Calif., USA).The Thielavia terrestris feruloyl esterase gene insert in pDFng106 wasconfirmed by DNA sequencing. A clone containing pDFng106 was picked intoeight 14 ml polypropylene round-bottom tubes each containing 3 ml of LBmedium supplemented with 100 μg of ampicillin per ml and grown overnightin at 37° C. and with shaking at 200 rpm. Plasmid pDFng106 was isolatedusing a QIAGEN® Mini Kit according to the manufacturer's instructions inpreparation for transforming Aspergillus niger MBin120 protoplasts.

The same 1.8 kb PCR fragment was cloned into pCR®2.1-TOPO® vector(Invitrogen, Carlsbad, Calif., USA) using a TOPO® TA CLONING® Kit(Invitrogen, Carlsbad, Calif., USA), to generate pDFng105 (FIG. 3). TheThielavia terrestris feruloyl esterase gene insert in pDFng105 wasconfirmed by DNA sequencing. E. coli pDFng105 was deposited with theAgricultural Research Service Patent Culture Collection, NorthernRegional Research Center, Peoria, Ill., USA, on Nov. 26, 2008 as NRRLB-50204.

Example 5 Characterization of the Thielavia terrestris Genomic SequenceEncoding a Feruloyl Esterase

DNA sequencing of the 1.8 kb PCR fragment was performed with aPerkin-Elmer Applied Biosystems Model 377 XL Automated DNA Sequencer(Perkin-Elmer/Applied Biosystems, Inc., Foster City, Calif., USA) usingdye-terminator chemistry (Giesecke et al., 1992, Journal of VirologyMethods 38: 47-60). The following primers were used for sequencing:

pDFng105 sequencing primers: M13 (−20) F: (SEQ ID NO: 6)5′-GTAAAACGACGGCCAGT-3′ M13 (−48) R: (SEQ ID NO: 7)5′-AGCGGATAACAATTTGACACAGGA-3′ pDFng106 sequencing primers: Na2Tpi F:(SEQ ID NO: 8) 5′-ACTCAATTTACCTCTATCCACACTT-3′ TtFaeCseqF:(SEQ ID NO: 9) 5′-AAGTCCTCACCAAGGGTTTC-3′ AMG R: (SEQ ID NO: 10)5′-CTATAGCGAAATGGATTGATTGTCT-3′

Nucleotide sequence data were scrutinized for quality and all sequenceswere compared to each other with assistance of PHRED/PHRAP software(University of Washington, Seattle, Wash., USA).

A gene model for the Thielavia terrestris feruloyl esterase sequence onscaffold 7 was constructed based on similarity of the encoded protein toknown homologs of feruloyl esterases. The nucleotide sequence (SEQ IDNO: 1) and deduced amino acid sequence (SEQ ID NO: 2) are shown inFIG. 1. The genomic fragment of 1824 bp (including the stop codon)encodes a polypeptide of 585 amino acids, interrupted by 1 predictedintron of 66 bp. The % G+C content of the gene and the mature codingsequence are 60.3% and 60.4%, respectively. Using the SignalP softwareprogram (Nielsen et al., 1997, Protein Engineering 10:1-6), a signalpeptide of 25 residues was predicted. The predicted mature proteincontains 560 amino acids with a molecular mass of 59.9 kDa.

A comparative pairwise global alignment of amino acid sequences wasdetermined using the Needleman-Wunsch algorithm (Needleman and Wunsch,1970, J. Mol. Biol. 48: 443-453) as implemented in the Needle program ofEMBOSS with gap open penalty of 10, gap extension penalty of 0.5, andthe EBLOSUM62 matrix. The alignment showed that the deduced amino acidsequence of the Thielavia terrestris gene encoding the maturepolypeptide having feruloyl esterase shared 75% identity (excludinggaps) to the deduced amino acid sequence of a tannase and feruloylesterase family protein from Neosartorya fischeri (UniProt Accessionnumber A1D9T4).

DEPOSIT OF BIOLOGICAL MATERIAL

The following biological material has been deposited under the terms ofthe Budapest Treaty with the Agricultural Research Service PatentCulture Collection (NRRL), Northern Regional Research Center, 1815University Street, Peoria, Ill., USA, and given the following accessionnumber:

Deposit Accession Number Date of Deposit E. coli pDFng105 NRRL B-50204Nov. 26, 2008

The strain has been deposited under conditions that assure that accessto the culture will be available during the pendency of this patentapplication to one determined by foreign patent laws to be entitledthereto. The deposit represents a substantially pure culture of thedeposited strain. The deposit is available as required by foreign patentlaws in countries wherein counterparts of the subject application, orits progeny are filed. However, it should be understood that theavailability of a deposit does not constitute a license to practice thesubject invention in derogation of patent rights granted by governmentalaction.

The present invention is further described by the following numberedparagraphs:

[1] An isolated polypeptide having feruloyl esterase activity, selectedfrom the group consisting of: (a) a polypeptide comprising an amino acidsequence having at least 75% sequence identity to the mature polypeptideof SEQ ID NO: 2; (b) a polypeptide encoded by a polynucleotide thathybridizes under at least medium-high stringency conditions with (i) themature polypeptide coding sequence of SEQ ID NO: 1, (ii) the cDNAsequence contained in the mature polypeptide coding sequence of SEQ IDNO: 1, or (iii) a full-length complementary strand of (i) or (ii); (c) apolypeptide encoded by a polynucleotide comprising a nucleotide sequencehaving at least 75% sequence identity to the mature polypeptide codingsequence of SEQ ID NO: 1; and (d) a variant comprising a substitution,deletion, and/or insertion of one or more (several) amino acids of themature polypeptide of SEQ ID NO: 2.

[2] The polypeptide of paragraph 1, comprising an amino acid sequencehaving at least 75% sequence identity to the mature polypeptide of SEQID NO: 2.

[3] The polypeptide of paragraph 2, comprising an amino acid sequencehaving at least 80% sequence identity to the mature polypeptide of SEQID NO: 2.

[4] The polypeptide of paragraph 3, comprising an amino acid sequencehaving at least 85% sequence identity to the mature polypeptide of SEQID NO: 2.

[5] The polypeptide of paragraph 4, comprising an amino acid sequencehaving at least 90% sequence identity to the mature polypeptide of SEQID NO: 2.

[6] The polypeptide of paragraph 5, comprising an amino acid sequencehaving at least 95% sequence identity to the mature polypeptide of SEQID NO: 2.

[7] The polypeptide of paragraph 6, comprising an amino acid sequencehaving at least 97% sequence identity to the mature polypeptide of SEQID NO: 2.

[8] The polypeptide of paragraph 1, comprising or consisting of theamino acid sequence of SEQ ID NO: 2; or a fragment thereof havingferuloyl esterase activity.

[9] The polypeptide of paragraph 8, comprising or consisting of theamino acid sequence of SEQ ID NO: 2.

[10] The polypeptide of paragraph 1, comprising or consisting of themature polypeptide of SEQ ID NO: 2.

[11] The polypeptide of paragraph 1, which is encoded by apolynucleotide that hybridizes under at least medium-high stringencyconditions with (i) the mature polypeptide coding sequence of SEQ ID NO:1, (ii) the cDNA sequence contained in the mature polypeptide codingsequence of SEQ ID NO: 1, or (iii) a full-length complementary strand of(i) or (ii).

[12] The polypeptide of paragraph 11, which is encoded by apolynucleotide that hybridizes under at least high stringency conditionswith (i) the mature polypeptide coding sequence of SEQ ID NO: 1, (ii)the cDNA sequence contained in the mature polypeptide coding sequence ofSEQ ID NO: 1, or (iii) a full-length complementary strand of (i) or(ii).

[13] The polypeptide of paragraph 12, which is encoded by apolynucleotide that hybridizes under at least very high stringencyconditions with (i) the mature polypeptide coding sequence of SEQ ID NO:1, (ii) the cDNA sequence contained in the mature polypeptide codingsequence of SEQ ID NO: 1, or (iii) a full-length complementary strand of(i) or (ii).

[14] The polypeptide of paragraph 1, which is encoded by apolynucleotide comprising a nucleotide sequence having at least 75%sequence identity to the mature polypeptide coding sequence of SEQ IDNO: 1.

[15] The polypeptide of paragraph 14, which is encoded by apolynucleotide comprising a nucleotide sequence having at least 80%sequence identity to the mature polypeptide coding sequence of SEQ IDNO: 1.

[16] The polypeptide of paragraph 15, which is encoded by apolynucleotide comprising a nucleotide sequence having at least 85%sequence identity to the mature polypeptide coding sequence of SEQ IDNO: 1.

[17] The polypeptide of paragraph 16, which is encoded by apolynucleotide comprising a nucleotide sequence having at least 90%sequence identity to the mature polypeptide coding sequence of SEQ IDNO: 1.

[18] The polypeptide of paragraph 17, which is encoded by apolynucleotide comprising a nucleotide sequence having at least 95%sequence identity to the mature polypeptide coding sequence of SEQ IDNO: 1.

[19] The polypeptide of paragraph 18, which is encoded by apolynucleotide comprising a nucleotide sequence having at least 97%sequence identity to the mature polypeptide coding sequence of SEQ IDNO: 1.

[20] The polypeptide of paragraph 1, which is encoded by apolynucleotide comprising or consisting of the nucleotide sequence ofSEQ ID NO: 1; or a subsequence thereof encoding a fragment havingferuloyl esterase activity.

[21] The polypeptide of paragraph 20, which is encoded by apolynucleotide comprising or consisting of the nucleotide sequence ofSEQ ID NO: 1.

[22] The polypeptide of paragraph 1, which is encoded by apolynucleotide comprising or consisting of the mature polypeptide codingsequence of SEQ ID NO: 1.

[23] The polypeptide of paragraph 1, wherein the polypeptide is avariant comprising a substitution, deletion, and/or insertion of one ormore (several) amino acids of the mature polypeptide of SEQ ID NO: 2.

[24] The polypeptide of paragraph 1, which is encoded by thepolynucleotide contained in plasmid pDFng105 which is contained in E.coli NRRL B-50204.

[25] The polypeptide of any of paragraphs 1-24, wherein the maturepolypeptide is amino acids 26 to 585 of SEQ ID NO: 2.

[26] The polypeptide of any of paragraphs 1-25, wherein the maturepolypeptide coding sequence is nucleotides 76 to 1821 of SEQ ID NO: 1.

[27] An isolated polynucleotide comprising a nucleotide sequence thatencodes the polypeptide of any of paragraphs 1-26.

[28] The isolated polynucleotide of paragraph 27, comprising at leastone mutation in the mature polypeptide coding sequence of SEQ ID NO: 1,in which the mutant nucleotide sequence encodes the mature polypeptideof SEQ ID NO: 2.

[29] A nucleic acid construct comprising the polynucleotide of paragraph27 or 28 operably linked to one or more (several) control sequences thatdirect the production of the polypeptide in an expression host.

[30] A recombinant expression vector comprising the polynucleotide ofparagraph 27 or 28.

[31] A recombinant host cell comprising the polynucleotide of paragraph27 or 28 operably linked to one or more (several) control sequences thatdirect the production of a polypeptide having alpha-glucuronidaseactivity.

[32] A method of producing the polypeptide of any of paragraphs 1-26,comprising: (a) cultivating a cell, which in its wild-type form producesthe polypeptide, under conditions conducive for production of thepolypeptide; and (b) recovering the polypeptide.

[33] A method of producing the polypeptide of any of paragraphs 1-26,comprising: (a) cultivating a host cell comprising a nucleic acidconstruct comprising a nucleotide sequence encoding the polypeptideunder conditions conducive for production of the polypeptide; and (b)recovering the polypeptide.

[34] A method of producing a mutant of a parent cell, comprisingdisrupting or deleting a polynucleotide encoding the polypeptide, or aportion thereof, of any of paragraphs 1-26, which results in the mutantproducing less of the polypeptide than the parent cell.

[35] A mutant cell produced by the method of paragraph 34.

[36] The mutant cell of paragraph 35, further comprising a gene encodinga native or heterologous protein.

[37] A method of producing a protein, comprising: (a) cultivating themutant cell of paragraph 36 under conditions conducive for production ofthe protein; and (b) recovering the protein.

[38] The isolated polynucleotide of paragraph 27 or 28, obtained by (a)hybridizing a population of DNA under at least medium-high stringencyconditions with (i) the mature polypeptide coding sequence of SEQ ID NO:1, (ii) the cDNA sequence contained in the mature polypeptide codingsequence of SEQ ID NO: 1, or (iii) a full-length complementary strand of(i) or (ii); and (b) isolating the hybridizing polynucleotide, whichencodes a polypeptide having feruloyl esterase activity.

[39] The isolated polynucleotide of paragraph 38, obtained by (a)hybridizing a population of DNA under at least high stringencyconditions with (i) the mature polypeptide coding sequence of SEQ ID NO:1, (ii) the cDNA sequence contained in the mature polypeptide codingsequence of SEQ ID NO: 1, or (iii) a full-length complementary strand of(i) or (ii); and (b) isolating the hybridizing polynucleotide, whichencodes a polypeptide having feruloyl esterase activity.

[40] The isolated polynucleotide of paragraph 39, obtained by (a)hybridizing a population of DNA under at least very high stringencyconditions with (i) the mature polypeptide coding sequence of SEQ ID NO:1, (ii) the cDNA sequence contained in the mature polypeptide codingsequence of SEQ ID NO: 1, or (iii) a full-length complementary strand of(i) or (ii); and (b) isolating the hybridizing polynucleotide, whichencodes a polypeptide having feruloyl esterase activity.

[41] The isolated polynucleotide of any of paragraphs 38-40, wherein themature polypeptide coding sequence is nucleotides 76 to 1821 of SEQ IDNO: 1.

[42] A method of producing a polynucleotide comprising a mutantnucleotide sequence encoding a polypeptide having feruloyl esteraseactivity, comprising: (a) introducing at least one mutation into themature polypeptide coding sequence of SEQ ID NO: 1, wherein the mutantnucleotide sequence encodes a polypeptide comprising or consisting ofthe mature polypeptide of SEQ ID NO: 2; and (b) recovering thepolynucleotide comprising the mutant nucleotide sequence.

[43] A mutant polynucleotide produced by the method of paragraph 42.

[44] A method of producing a polypeptide, comprising: (a) cultivating acell comprising the mutant polynucleotide of paragraph 43 encoding thepolypeptide under conditions conducive for production of thepolypeptide; and (b) recovering the polypeptide.

[45] A method of producing the polypeptide of any of paragraphs 1-26,comprising: (a) cultivating a transgenic plant or a plant cellcomprising a polynucleotide encoding the polypeptide under conditionsconducive for production of the polypeptide; and (b) recovering thepolypeptide.

[46] A transgenic plant, plant part or plant cell transformed with apolynucleotide encoding the polypeptide of any of paragraphs 1-26.

[47] A double-stranded inhibitory RNA (dsRNA) molecule comprising asubsequence of the polynucleotide of paragraph 27 or 28, whereinoptionally the dsRNA is a siRNA or a miRNA molecule.

[48] The double-stranded inhibitory RNA (dsRNA) molecule of paragraph47, which is about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or moreduplex nucleotides in length.

[49] A method of inhibiting the expression of a polypeptide havingferuloyl esterase activity in a cell, comprising administering to thecell or expressing in the cell a double-stranded RNA (dsRNA) molecule,wherein the dsRNA comprises a subsequence of the polynucleotide ofparagraph 27 or 28.

[50] The method of paragraph 49, wherein the dsRNA is about 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25 or more duplex nucleotides in length.

[51] An isolated polynucleotide encoding a signal peptide comprising orconsisting of amino acids 1 to 25 of SEQ ID NO: 2.

[52] A nucleic acid construct comprising a gene encoding a proteinoperably linked to the polynucleotide of paragraph 51, wherein the geneis foreign to the polynucleotide encoding the signal peptide.

[53] A recombinant expression vector comprising a gene encoding aprotein operably linked to the polynucleotide of paragraph 51, whereinthe gene is foreign to the polynucleotide encoding the signal peptide.

[54] A recombinant host cell comprising a gene encoding a proteinoperably linked to the polynucleotide of paragraph 51, wherein the geneis foreign to the polynucleotide encoding the signal peptide.

[55] A method of producing a protein, comprising: (a) cultivating arecombinant host cell comprising a gene encoding a protein operablylinked to the polynucleotide of paragraph 51, wherein the gene isforeign to the polynucleotide encoding the signal peptide, underconditions conducive for production of the protein; and (b) recoveringthe protein.

[56] A composition comprising the polypeptide of any of paragraphs 1-26.

[57] A method for degrading or converting a cellulosic material,comprising: treating the cellulosic material with an enzyme compositionin the presence of the polypeptide having feruloyl esterase activity ofany of paragraphs 1-26.

[58] The method of paragraph 57, wherein the cellulosic material ispretreated.

[59] The method of paragraph 57 or 58, wherein the enzyme compositioncomprises one or more (several) cellulolytic enzymes selected from thegroup consisting of an endoglucanase, a cellobiohydrolase, and abeta-glucosidase.

[60] The method of any of paragraphs 57-59, wherein the enzymecomposition further comprises a polypeptide having cellulolyticenhancing activity.

[61] The method of any of paragraphs 57-60, wherein the enzymecomposition further comprises one or more (several) enzymes selectedfrom the group consisting of a hemicellulase, an esterase, a protease, alaccase, and a peroxidase.

[62] The method of any of paragraphs 57-61, wherein the enzymecomposition further comprises one or more (several) enzymes selectedfrom the group consisting of a xylanase, an acetyxylan esterase, aferuloyl esterase, an arabinofuranosidase, a xylosidase, aglucuronidase, and a combination thereof.

[63] The method of any of paragraphs 57-62, further comprisingrecovering the degraded cellulosic material.

[64] The method of paragraph 63, wherein the degraded cellulosicmaterial is a sugar.

[65] The method of paragraph 64, wherein the sugar is selected from thegroup consisting of glucose, xylose, mannose, galactose, and arabinose.

[66] A method for producing a fermentation product, comprising: (a)saccharifying a cellulosic material with an enzyme composition in thepresence of the polypeptide having feruloyl esterase activity of any ofparagraphs 1-26; (b) fermenting the saccharified cellulosic materialwith one or more (several) fermenting microorganisms to produce thefermentation product; and (c) recovering the fermentation product fromthe fermentation.

[67] The method of paragraph 66, wherein the cellulosic material ispretreated.

[68] The method of paragraph 66 or 67, wherein the enzyme compositioncomprises one or more (several) cellulolytic enzymes selected from thegroup consisting of an endoglucanase, a cellobiohydrolase, and abeta-glucosidase.

[69] The method of any of paragraphs 66-68, wherein the enzymecomposition further comprises a polypeptide having cellulolyticenhancing activity.

[70] The method of any of paragraphs 66-69, wherein the enzymecomposition further comprises one or more (several) enzymes selectedfrom the group consisting of a hemicellulase, an esterase, a protease, alaccase, and a peroxidase.

[71] The method of any of paragraphs 66-70, wherein the enzymecomposition further comprises one or more (several) enzymes selectedfrom the group consisting of a xylanase, an acetyxylan esterase, aferuloyl esterase, an arabinofuranosidase, a xylosidase, aglucuronidase, and a combination thereof.

[72] The method of any of paragraphs 66-71, wherein steps (a) and (b)are performed simultaneously in a simultaneous saccharification andfermentation.

[73] The method of any of paragraphs 66-72, wherein the fermentationproduct is an alcohol, an organic acid, a ketone, an amino acid, or agas.

[74] A method of fermenting a cellulosic material, comprising:fermenting the cellulosic material with one or more (several) fermentingmicroorganisms, wherein the cellulosic material is saccharified with anenzyme composition in the presence of the polypeptide having feruloylesterase activity of any of paragraphs 1-26.

[75] The method of paragraph 74, wherein the fermenting of thecellulosic material produces a fermentation product.

[76] The method of paragraph 75, further comprising recovering thefermentation product from the fermentation.

[77] The method of any of paragraphs 74-76, wherein the cellulosicmaterial is pretreated before saccharification.

[78] The method of any of paragraphs 74-77, wherein the enzymecomposition comprises one or more (several) cellulolytic enzymesselected from the group consisting of an endoglucanase, acellobiohydrolase, and a beta-glucosidase.

[79] The method of any of paragraphs 74-78, wherein the enzymecomposition further comprises a polypeptide having cellulolyticenhancing activity.

[80] The method of any of paragraphs 74-78, wherein the enzymecomposition further comprises one or more (several) enzymes selectedfrom the group consisting of a hemicellulase, an esterase, a protease, alaccase, and a peroxidase.

[81] The method of any of paragraphs 74-80, wherein the enzymecomposition further comprises one or more (several) enzymes selectedfrom the group consisting of a xylanase, an acetyxylan esterase, aferuloyl esterase, an arabinofuranosidase, a xylosidase, aglucuronidase, and a combination thereof.

[82] The method of any of paragraphs 74-81, wherein the fermentationproduct is an alcohol, an organic acid, a ketone, an amino acid, or agas.

[83] A method for degrading or converting a xylan-containing material,comprising: treating the xylan-containing material with an enzymecomposition in the presence of the polypeptide having feruloyl esteraseactivity of any of paragraphs 1-26.

[84] The method of paragraph 83, wherein the xylan-containing materialis pretreated.

[85] The method of paragraph 83 or 84, wherein the enzyme compositioncomprises one or more (several) enzymes selected from the groupconsisting of a xylanase, an acetyxylan esterase, a feruloyl esterase,an arabinofuranosidase, a xylosidase, a glucuronidase, and a combinationthereof.

[86] The method of any of paragraphs 83-85, wherein the enzymecomposition further comprises one or more (several) enzymes selectedfrom the group consisting of a hemicellulase, an esterase, a protease, alaccase, and a peroxidase.

[87] The method of any of paragraphs 83-86, wherein the enzymecomposition further comprises one or more (several) enzymes selectedfrom the group consisting of an endoglucanase, a cellobiohydrolase, anda beta-glucosidase.

[88] The method of paragraph 87, wherein the enzyme composition furthercomprises a polypeptide having cellulolytic enhancing activity.

[89] The method of any of paragraphs 83-88, further comprisingrecovering the degraded xylan-containing material.

[90] A method of producing a fermentation product, comprising: (a)saccharifying a xylan-containing material with an enzyme composition inthe presence of a polypeptide having feruloyl esterase activity of anyof paragraphs 1-26; (b) fermenting the saccharified xylan-containingmaterial with one or more (several) fermenting microorganisms to producethe fermentation product; and (c) recovering the fermentation productfrom the fermentation.

[91] The method of paragraph 90, wherein the xylan-containing materialis pretreated.

[92] The method of paragraph 90 or 91, wherein the enzyme compositioncomprises one or more (several) enzymes selected from the groupconsisting of a xylanase, an acetyxylan esterase, a feruloyl esterase,an arabinofuranosidase, a xylosidase, a glucuronidase, and a combinationthereof.

[93] The method of any of paragraphs 90-92 wherein the enzymecomposition further comprises one or more (several) enzymes selectedfrom the group consisting of a hemicellulase, an esterase, a protease, alaccase, and a peroxidase.

[94] The method of any of paragraphs 90-94, wherein the enzymecomposition further comprises one or more (several) enzymes selectedfrom the group consisting of an endoglucanase, a cellobiohydrolase, anda beta-glucosidase.

[95] The method of paragraph 94, further comprising a polypeptide havingcellulolytic enhancing activity.

[96] The method of any of paragraphs 90-99, wherein steps (a) and (b)are performed simultaneously in a simultaneous saccharification andfermentation.

[97] The method of any of paragraphs 90-96, wherein the fermentationproduct is an alcohol, an organic acid, a ketone, an amino acid, or agas.

[98] A method of fermenting a xylan-containing material, comprising:fermenting the xylan-containing material with one or more (several)fermenting microorganisms, wherein the xylan-containing material issaccharified with an enzyme composition in the presence of thepolypeptide having feruloyl esterase activity of any of paragraphs 1-26.

[99] The method of paragraph 98, wherein the fermenting of thexylan-containing material produces a fermentation product.

[100] The method of paragraph 99, further comprising recovering thefermentation product from the fermentation.

[101] The method of any of paragraphs 98-100, wherein thexylan-containing material is pretreated before saccharification.

[102] The method of any of paragraphs 98-101, wherein the enzymecomposition comprises one or more (several) enzymes selected from thegroup consisting of a xylanase, an acetyxylan esterase, a feruloylesterase, an arabinofuranosidase, a xylosidase, a glucuronidase, and acombination thereof.

[103] The method of any of paragraphs 98-102 wherein the enzymecomposition further comprises one or more (several) enzymes selectedfrom the group consisting of a hemicellulase, an esterase, a protease, alaccase, and a peroxidase.

[104] The method of any of paragraphs 98-103, wherein the enzymecomposition further comprises one or more (several) enzymes selectedfrom the group consisting of an endoglucanase, a cellobiohydrolase, anda beta-glucosidase.

[105] The method of paragraph 104, wherein the enzyme compositionfurther comprises a polypeptide having cellulolytic enhancing activity.

[106] The method of any of paragraphs 98-105, wherein the fermentationproduct is an alcohol, an organic acid, a ketone, an amino acid, or agas.

[107] A method of fermenting a xylan-containing material, comprising:fermenting the xylan-containing material with one or more (several)fermenting microorganisms, wherein the xylan-containing material issaccharified with an enzyme composition in the presence of thepolypeptide having feruloyl esterase activity of any of paragraphs 1-26.

[108] The method of paragraph 107, wherein the fermenting of thexylan-containing material produces a fermentation product.

[109] The method of paragraph 108, further comprising recovering thefermentation product from the fermentation.

[110] The method of any of paragraphs 107-109, wherein thexylan-containing material is pretreated before saccharification.

[111] The method of any of paragraphs 107-110, wherein the enzymecomposition further comprises one or more (several) enzymes selectedfrom the group consisting of a xylanase, an acetyxylan esterase, aferuloyl esterase, an arabinofuranosidase, a xylosidase, aglucuronidase, and a combination thereof.

[112] The method of any of paragraphs 107-111, wherein the enzymecomposition further comprises one or more (several) enzymes selectedfrom the group consisting of a hemicellulase, an esterase, a protease, alaccase, and a peroxidase.

[113] The method of any of paragraphs 107-112, wherein the enzymecomposition comprises one or more (several) cellulolytic enzymesselected from the group consisting of an endoglucanase, acellobiohydrolase, and a beta-glucosidase.

[114] The method of paragraph 113, wherein the enzyme compositionfurther comprises a polypeptide having cellulolytic enhancing activity.

[115] The method of any of paragraphs 107-114, wherein the fermentationproduct is an alcohol, an organic acid, a ketone, an amino acid, or agas.

The invention described and claimed herein is not to be limited in scopeby the specific aspects herein disclosed, since these aspects areintended as illustrations of several aspects of the invention. Anyequivalent aspects are intended to be within the scope of thisinvention. Indeed, various modifications of the invention in addition tothose shown and described herein will become apparent to those skilledin the art from the foregoing description. Such modifications are alsointended to fall within the scope of the appended claims. In the case ofconflict, the present disclosure including definitions will control.

1. An isolated polypeptide having feruloyl esterase activity, selected from the group consisting of: (a) a polypeptide comprising an amino acid sequence having at least 75% sequence identity to the mature polypeptide of SEQ ID NO: 2; (b) a polypeptide encoded by a polynucleotide that hybridizes under at least medium-high stringency conditions with (i) the mature polypeptide coding sequence of SEQ ID NO: 1, (ii) the cDNA sequence contained in the mature polypeptide coding sequence of SEQ ID NO: 1, or (iii) a full-length complementary strand of (i) or (ii); (c) a polypeptide encoded by a polynucleotide comprising a nucleotide sequence having at least 75% sequence identity to the mature polypeptide coding sequence of SEQ ID NO: 1; and (d) a variant comprising a substitution, deletion, and/or insertion of one or more (several) amino acids of the mature polypeptide of SEQ ID NO:
 2. 2. The polypeptide of claim 1, comprising or consisting of the mature polypeptide of SEQ ID NO:
 2. 3. The polypeptide of claim 1, which is encoded by the polynucleotide contained in plasmid pDFng105 which is contained in E. coli NRRL B-50204.
 4. An isolated polynucleotide comprising a nucleotide sequence that encodes the polypeptide of claim
 1. 5. A recombinant host cell comprising the polynucleotide of claim 4 operably linked to one or more (several) control sequences that direct the production of a polypeptide having alpha-glucuronidase activity.
 6. A method of producing the polypeptide of claim 1, comprising: (a) cultivating a cell, which in its wild-type form produces the polypeptide, under conditions conducive for production of the polypeptide; and (b) recovering the polypeptide.
 7. A method of producing the polypeptide of claim 1, comprising: (a) cultivating a host cell comprising a nucleic acid construct comprising a nucleotide sequence encoding the polypeptide under conditions conducive for production of the polypeptide; and (b) recovering the polypeptide.
 8. A method of producing a mutant of a parent cell, comprising disrupting or deleting a polynucleotide encoding the polypeptide, or a portion thereof, of claim 1, which results in the mutant producing less of the polypeptide than the parent cell.
 9. A method of producing the polypeptide of claim 1, comprising: (a) cultivating a transgenic plant or a plant cell comprising a polynucleotide encoding the polypeptide under conditions conducive for production of the polypeptide; and (b) recovering the polypeptide.
 10. A transgenic plant, plant part or plant cell transformed with a polynucleotide encoding the polypeptide of claim
 1. 11. A double-stranded inhibitory RNA (dsRNA) molecule comprising a subsequence of the polynucleotide of claim 4, wherein optionally the dsRNA is a siRNA or a miRNA molecule.
 12. A method of inhibiting the expression of a polypeptide having feruloyl esterase activity in a cell, comprising administering to the cell or expressing in the cell a double-stranded RNA (dsRNA) molecule, wherein the dsRNA comprises a subsequence of the polynucleotide of claim
 4. 13. An isolated polynucleotide encoding a signal peptide comprising or consisting of amino acids 1 to 25 of SEQ ID NO:
 2. 14. A method of producing a protein, comprising: (a) cultivating a recombinant host cell comprising a gene encoding a protein operably linked to the polynucleotide of claim 13, wherein the gene is foreign to the polynucleotide encoding the signal peptide, under conditions conducive for production of the protein; and (b) recovering the protein.
 15. A method for degrading or converting a xylan-containing material or cellulosic material, comprising: treating the xylan-containing material or cellulosic material with an enzyme composition in the presence of the polypeptide having feruloyl esterase activity of claim
 1. 16. (canceled)
 17. A method of producing a fermentation product, comprising: (a) saccharifying a xylan-containing material or cellulosic material with an enzyme composition in the presence of a polypeptide having feruloyl esterase activity of claim 1; (b) fermenting the saccharified xylan-containing material or cellulosic material with one or more (several) fermenting microorganisms to produce the fermentation product; and (c) recovering the fermentation product from the fermentation.
 18. A method of fermenting a xylan-containing material or cellulosic material, comprising: fermenting the xylan-containing material or cellulosic material with one or more (several) fermenting microorganisms, wherein the xylan-containing material or cellulosic material is saccharified with an enzyme composition in the presence of the polypeptide having feruloyl esterase activity of claim
 1. 19. (canceled)
 20. (canceled)
 21. A method of fermenting a xylan-containing material or cellulosic material, comprising: fermenting the xylan-containing material or cellulosic material with one or more (several) fermenting microorganisms, wherein the xylan-containing material or cellulosic material is saccharified with an enzyme composition in the presence of the polypeptide having feruloyl esterase activity of claim
 1. 22. The method of claim 21, wherein the fermenting of the xylan-containing material or cellulosic material produces a fermentation product.
 23. The method of claim 22, further comprising recovering the fermentation product from the fermentation.
 24. (canceled)
 25. (canceled) 